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Currently, the majority of patients with acute myeloid leukemia (AML) still die of their disease due to primary resistance or relapse toward conventional reactive oxygen species (ROS)- and DNA damage–inducing chemotherapy regimens. Herein, we explored the therapeutic potential to enhance chemotherapy response in AML, by targeting the ROS scavenger enzyme MutT homolog 1 (MTH1, NUDT1), which protects cellular integrity through prevention of fatal chemotherapy-induced oxidative DNA damage. We demonstrate that MTH1 is a potential druggable target expressed by the majority of patients with AML and the inv(16)/KITD816Y AML mouse model mimicking the genetics of patients with AML exhibiting poor response to standard chemotherapy (i.e., anthracycline & cytarabine). Strikingly, combinatorial treatment of inv(16)/KITD816Y AML cells with the MTH1 inhibitor TH1579 and ROS- and DNA damage-inducing standard chemotherapy induced growth arrest and incorporated oxidized nucleotides into DNA leading to significantly increased DNA damage. Consistently, TH1579 and chemotherapy synergistically inhibited growth of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic bone marrow cells. In addition, combinatorial treatment of inv(16)/KITD816Y AML mice with TH1579 and chemotherapy significantly reduced AML burden and prolonged survival compared with untreated or single treated mice. In conclusion, our study provides a rationale for future clinical studies combining standard AML chemotherapy with TH1579 to boost standard chemotherapy response in patients with AML. Moreover, other cancer entities treated with ROS- and DNA damage–inducing chemo- or radiotherapies might benefit therapeutically from complementary treatment with TH1579.

Supplementary Figure from Inhibition of Oxidized Nucleotide Sanitation By TH1579 and Conventional Chemotherapy Cooperatively Enhance Oxidative DNA Damage and Survival in AML

Supplementary figures and tables and material and methods

Supplementary Data from Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD–Driven AML

The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases. SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its autoinhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations. In this study, we established cells with acquired resistance to the allosteric SHP2 inhibitor SHP099 from two FLT3-ITD (internal tandem duplication)-positive AML cell lines. Label-free and isobaric labeling quantitative mass spectrometry–based phosphoproteomics of these resistant models demonstrated that AML cells can restore phosphorylated ERK (pERK) in the presence of SHP099, thus developing adaptive resistance. Mechanistically, SHP2 inhibition induced tyrosine phosphorylation and feedback-driven activation of the FLT3 receptor, which in turn phosphorylated SHP2 on tyrosine 62. This phosphorylation stabilized SHP2 in its open conformation, preventing SHP099 binding and conferring resistance. Combinatorial inhibition of SHP2 and MEK or FLT3 prevented pERK rebound and resistant cell growth. The same mechanism was observed in a FLT3-mutated B-cell acute lymphoblastic leukemia cell line and in the inv(16)/KitD816Y AML mouse model, but allosteric inhibition of Shp2 did not impair the clonogenic ability of normal bone marrow progenitors. Together, these results support the future use of SHP2 inhibitor combinations for clinical applications.Significance:These findings suggest that combined inhibition of SHP2 and FLT3 effectively treat FLT3-ITD–positive AML, highlighting the need for development of more potent SHP2 inhibitors and combination therapies for clinical applications.

Supplementary Figure from Phosphorylation of SHP2 at Tyr62 Enables Acquired Resistance to SHP2 Allosteric Inhibitors in FLT3-ITD–Driven AML

Funding Information: This study was supported by the Cancer Foundation Finland, the Helsinki Institute of Life Science (HiLIFE) Fellow grants, the Academy of Finland (grant 1320185 ), the Sigrid Jusélius Foundation , the Finnish Cancer Institute , the Finnish Medical Foundation , University of Helsinki , and the Finnish special governmental subsidy for health sciences, research, and training . The FIMM High Throughput Biomedicine Unit is financially supported by the University of Helsinki (HiLIFE) and Biocenter Finland. O.D. was supported by the K. Albin Johansson Foundation , the Emil Aaltonen Foundation , and the Juhani Aho foundation . Publisher Copyright: © 2023 The American Society of Hematology Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases. SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its autoinhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations. In this study, we established cells with acquired resistance to the allosteric SHP2 inhibitor SHP099 from two FLT3-ITD (internal tandem duplication)-positive AML cell lines. Label-free and isobaric labeling quantitative mass spectrometry–based phosphoproteomics of these resistant models demonstrated that AML cells can restore phosphorylated ERK (pERK) in the presence of SHP099, thus developing adaptive resistance. Mechanistically, SHP2 inhibition induced tyrosine phosphorylation and feedback-driven activation of the FLT3 receptor, which in turn phosphorylated SHP2 on tyrosine 62. This phosphorylation stabilized SHP2 in its open conformation, preventing SHP099 binding and conferring resistance. Combinatorial inhibition of SHP2 and MEK or FLT3 prevented pERK rebound and resistant cell growth. The same mechanism was observed in a FLT3-mutated B-cell acute lymphoblastic leukemia cell line and in the inv(16)/KitD816Y AML mouse model, but allosteric inhibition of Shp2 did not impair the clonogenic ability of normal bone marrow progenitors. Together, these results support the future use of SHP2 inhibitor combinations for clinical applications. Significance: These findings suggest that combined inhibition of SHP2 and FLT3 effectively treat FLT3-ITD–positive AML, highlighting the need for development of more potent SHP2 inhibitors and combination therapies for clinical applications.

Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KITD816Y AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KITD816Y AML cells in response to chemotherapy. Consistently, combinations of chemotherapy and PI3K or DNA-PK inhibitors synergistically inhibited growth and survival of clonogenic AML cells without substantially inhibiting normal clonogenic bone marrow cells. Moreover, treatment of inv(16)/KITD816Y AML mice with combinations of chemotherapy and PI3K or DNA-PK inhibitors significantly prolonged survival compared to untreated/single-treated mice. Mechanistically, our findings implicate that constitutive activation of PI3K/AKT signaling driven by mutant KIT, and potentially other mutational activators such as FLT3 and RAS, cooperates with chemotherapy-induced DNA-PK-dependent activation of AKT to promote survival, DNA repair, and chemotherapy resistance in AML. Hence, our study provides a rationale to select AML patients exhibiting constitutive PI3K/AKT activation for simultaneous treatment with chemotherapy and inhibitors of DNA-PK and PI3K to improve chemotherapy response and clinical outcome.

Oda Jensen,1 Andreas Kiesbye Øvlisen,1,2 Lasse Hjort Jakobsen,1,2 Anne Stidsholt Roug,1,2 René Ernst Nielsen,2,3 Claus Werenberg Marcher,4 Lene Hyldahl Ebbesen,5 Kim Theilgaard-Mönch,6 Peter Møller,7 Claudia Schöllkopf,8 Christian Torp-Pedersen,9,10 Tarec Christoffer El-Galaly,1,2 Marianne Tang Severinsen1,2 1Department of Haematology, Clinical Cancer Research Centre, Aalborg University Hospital, Aalborg, Denmark; 2Department of Clinical Medicine, Aalborg University, Aalborg, Denmark; 3Department of Psychiatry, Aalborg University Hospital, Aalborg, Denmark; 4Department of Haematology, Odense University Hospital, Odense, Denmark; 5Department of Haematology, Aarhus University Hospital, Aarhus, Denmark; 6Department of Haematology, Rigshospitalet, Copenhagen, Denmark; 7Department of Haematology, Roskilde Sygehus, Roskilde, Denmark; 8Department of Haematology, Herlev Hospital, Herlev, Denmark; 9Unit of Clinical Biostatistics and Epidemiology, Aalborg University Hospital, Aalborg, Denmark; 10Department of Cardiology, Nordsjællands Hospital, Hillerød, DenmarkCorrespondence: Marianne Tang Severinsen, Department of Haematology, Clinical Cancer Research Centre, Aalborg University Hospital, Aalborg, Denmark, Email m.severinsen@rn.dkIntroduction: The diagnosis of a life-threatening disease can lead to depression and anxiety resulting in pharmacological treatment. However, use of psychotropic drugs (antidepressants, anxiolytics, and antipsychotics) in acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) is undetermined.Methods: Prescription of psychotropic drugs in Danish AML and MDS patients was compared to a cohort matched on age, sex, and country of origin from the Danish background population using national population-based registries.Results: In total, 2404 AML patients (median age 69 years) and 1307 MDS patients (median age 75 years) were included and each matched to five comparators from the background population. Two-year cumulative incidences showed that AML (20.6%) and MDS (21.2%) patients had a high risk of redemption of a psychotropic drug prescription compared to the background population (7.0% and 7.9%). High age, low educational level, and Charlson Comorbidity Index score ≥ 1 was associated with a higher risk in AML and MDS patients. Furthermore, non-curative treatment intent and performance status in AML patients, and high risk MDS were associated with elevated risk of psychotropic drug prescription.Conclusion: In conclusion, diagnoses of AML and MDS were associated with a higher rate of psychotropic drugs prescription compared to the background population.Keywords: acute myeloid leukaemia, myelodysplastic syndrome, depression, anxiety, psychotropic drugs

In the present study, we quantify the progress in overall survival (OS) during the period 2000-2016 among Danish patients with acute myeloid leukaemia (AML). This population-based study, including 3820 adult patients with AML, demonstrates a significantly improved OS over time with the 2-year age-standardised OS increasing from 22% in 2002 to 31% in 2016. The improvement in OS was exclusively seen in patients with AML aged ≥50 years, with absolute improvements in 2-year OS from 2002 to 2016 of ≥10% among patients aged 50-75 years and a small absolute increase in those aged >75 years.

Currently, the majority of patients with acute myeloid leukemia (AML) still die of their disease due to primary resistance or relapse toward conventional reactive oxygen species (ROS)- and DNA damage–inducing chemotherapy regimens. Herein, we explored the therapeutic potential to enhance chemotherapy response in AML, by targeting the ROS scavenger enzyme MutT homolog 1 (MTH1, NUDT1), which protects cellular integrity through prevention of fatal chemotherapy-induced oxidative DNA damage. We demonstrate that MTH1 is a potential druggable target expressed by the majority of patients with AML and the inv(16)/KITD816Y AML mouse model mimicking the genetics of patients with AML exhibiting poor response to standard chemotherapy (i.e., anthracycline & cytarabine). Strikingly, combinatorial treatment of inv(16)/KITD816Y AML cells with the MTH1 inhibitor TH1579 and ROS- and DNA damage-inducing standard chemotherapy induced growth arrest and incorporated oxidized nucleotides into DNA leading to significantly increased DNA damage. Consistently, TH1579 and chemotherapy synergistically inhibited growth of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic bone marrow cells. In addition, combinatorial treatment of inv(16)/KITD816Y AML mice with TH1579 and chemotherapy significantly reduced AML burden and prolonged survival compared with untreated or single treated mice. In conclusion, our study provides a rationale for future clinical studies combining standard AML chemotherapy with TH1579 to boost standard chemotherapy response in patients with AML. Moreover, other cancer entities treated with ROS- and DNA damage–inducing chemo- or radiotherapies might benefit therapeutically from complementary treatment with TH1579.

Resistance of cancer patients to DNA damaging radiation therapy and chemotherapy remains a major problem in the clinic. The current review discusses the molecular mechanisms of therapy resistance in acute myeloid leukemia (AML) conferred by cooperative chemotherapy-induced DNA damage response (DDR) and mutational activation of PI3K/AKT signaling. In addition, strategies to overcome resistance are discussed, with particular focus on studies underpinning the vast potential of therapies combining standard chemotherapy AML regimens with small molecule inhibitors targeting key regulatory hubs at the interface of DDR and oncogenic signaling pathways.

In a recent study by our group we showed a disappointing absence of survival improvement among elderly acute myeloid leukemia (AML) patients from 2001 until 2016. The importance of which is undersc...

Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements. Here the authors show that differentiation of haematopoietic stem cells into mature blood cells is primed by cell type-specific transcription factors at the enhancer level during early differentiation, before they confere promoter-driven growth arrest, and activate post-mitotic terminal differentiation.

Summary With rising life expectancy, the importance of patient‐related prognostic factors and how to integrate such data into clinical decision‐making becomes increasingly important. The aim of this study was to evaluate the prognostic impact of smoking status in patients with acute myeloid leukaemia (AML) treated with intensive chemotherapy. We conducted a nationwide cohort study based on data obtained from the Danish National Leukaemia Registry (DNLR). The study comprised Danish patients aged 18–75 years, diagnosed with AML between 1 January 2000 and 31 December 2012. Medical records were reviewed and data on smoking status were collected. A total of 1040 patients (median age 59 years) were included, and 602 patients (58·9%) were categorised as ever‐smokers and the remaining as never‐smokers. Kaplan–Meier survival estimates revealed that ever‐smokers had a significant shorter median overall survival (OS) at 17·2 months [95% CI (14·9;19·1)] compared to never‐smokers at 24·5 months (95% CI [19·2;30·7]). Multivariate analysis revealed smoking status as a significant prognostic factor for inferior OS with a hazard ratio (HR) of 1·22 [95% CI (1·04;1·44)]. In conclusion, smoking status was found to be associated with inferior OS in intensively treated AML patients.

Key Points EMCN is a novel marker of human HSCs. EMCN is a more specific marker of HSCs than CD34 as it can discriminate HSCs from lineage-committed HPCs.

Human myelopoiesis has been proposed to occur through oligopotent common myeloid progenitor (CMP) and lymphoid-primed multipotent progenitor (LMPP) populations. However, other studies have proposed direct commitment of multipotent cells to unilineage fates, without specific intermediary lineage cosegregation patterns. We here show that distinct human myeloid progenitor populations generate the neutrophil/monocyte and mast cell/basophil/eosinophil lineages as previously shown in mouse. Moreover, we find that neutrophil/monocyte potential selectively cosegregates with lymphoid lineage and mast cell/basophil/eosinophil potentials with megakaryocyte/erythroid potential early during lineage commitment. Furthermore, after this initial commitment step, mast cell/basophil/eosinophil and megakaryocyte/erythroid potentials colocalize at the single-cell level in restricted oligopotent progenitors. These results show that human myeloid lineages are generated through two distinct cellular pathways defined by complementary oligopotent cell populations.

In CEBPA-mutant leukemia, CEBPA up-regulates cancer-protective and -targetable CD73, indicating a novel potential therapy., The key myeloid transcription factor (TF), CEBPA, is frequently mutated in acute myeloid leukemia (AML), but the direct molecular effects of this leukemic driver mutation remain elusive. To investigate CEBPA mutant AML, we performed microscale, in vivo chromatin immunoprecipitation sequencing and identified a set of aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. Comparing gene expression changes in human CEBPA mutant AML and the corresponding CebpaLp30 mouse model, we identified Nt5e, encoding CD73, as a cross-species AML gene with an upstream leukemic enhancer physically and functionally linked to the gene. Increased expression of CD73, mediated by the CEBPA-p30 isoform, sustained leukemic growth via the CD73/A2AR axis. Notably, targeting of this pathway enhanced survival of AML-transplanted mice. Our data thus indicate a first-in-class link between a cancer driver mutation in a TF and a druggable, direct transcriptional target.

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients. © 2011 Macmillan Publishers Limited All rights reserved.

In this issue of Blood, Josefsdottir et al provide substantial evidence that commensal gut microbes regulate and sustain normal steady-state hematopoiesis.

The current study reports a flow cytometry-based protocol for the prospective purification of human BM populations representing six successive stages of terminal neutrophil differentiation, including early promyelocytes and late promyelocytes, myelocytes, metamyelocytes, band cells, and PMN neutrophilic granulocytes. Validation experiments revealed a high purity of each bone marrow population and biological meaningful expression profiles for marker genes of neutrophil differentiation at a hitherto unprecedented resolution. Hence, the present protocol should be useful for studying neutrophil differentiation in vivo in the human setting and constitutes an important alternative to models that are based on in vitro differentiation of myeloid cell lines and HPCs.

The secretory leukocyte protease inhibitor (SLPI) re-establishes homeostasis at sites of infection by virtue of its ability to exert antimicrobial activity, to suppress LPS-induced cellular immune responses, and to reduce tissue damage through inhibition of serine proteases released by polymorphonuclear neutrophil granulocytes (PMNs). Microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils demonstrates a transient, high mRNA expression of SLPI and genuine secondary granule proteins (GPs) in MYs. Consistent with this finding, immunostaining of BM cells showed SLPI and the secondary GP lactoferrin (LF) to be present in cells from the myelocyte stage and throughout neutrophil differentiation. Subcellular fractionation studies demonstrated the colocalization of SLPI and LF in subcellular fractions highly enriched in secondary granules. Finally, exocytosis studies demonstrated a corelease of SLPI and LF within minutes of activation. Collectively, these findings strongly indicate that SLPI is localized in secondary granules of PMNs. However, the amount of SLPI detected in PMNs is low compared with primary keratinocytes stimulated by growth factors involved in wound healing. This implicates that neutrophil-derived SLPI might not contribute essentially to re-establishment of homeostasis at sites of infection but rather, exert physiologically relevant intracellular activities. These might include the protection of secondary GPs against proteolytic activation and/or degradation by proteases, which might be dislocated to secondary granules at minute amounts as a consequence of spillover.

The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.

Systems biology has emerged as a new scientific field, which aims at investigating biological processes at the genomic and proteomic levels. Recent studies have unravelled aspects of neutrophil differentiation and immune responses at the systems level using high-throughput technologies. These studies have identified a plethora of novel effector proteins stored in the granules of neutrophils. In addition, these studies provide evidence that neutrophil differentiation and immune response are governed by a highly coordinated transcriptional programme that regulates cellular fate and function in a context-dependent manner.

The frequently detected t(4;14)(p16.3;q32) translocation in multiple myeloma (MM) results in a dysregulation of two potential oncogenes: multiple myeloma SET domain (MMSET) and fibroblast growth factor receptor 3 (FGFR3). As the expression of FGFR3 is undetectable in 30% of the t(4;14)+ MM patients, MMSET has been suggested to play an important role in the malignant transformation associated with the t(4;14) translocation. Screening with a real-time polymerase chain reaction (PCR) found complex expression patterns of the MMSET transcripts in fluorescence-activated cell sorted (FACS)-purified plasma cells (PCs) from 15 t(4;14)+ MM patients. In addition, potential target genes of MMSET type I and II were identified, using microarray analyses of MMSET transfected cell lines. Subsequently, the expression of potential target genes was verified by real-time PCR in FACS-purified PCs from 15 t(4;14)+ and 22 t(4;14)- MM patients. We suggest that the inhibitor of differentiation 1 (ID-1) is a target gene of MMSET, based on its upregulation in MMSET transfected cell lines and a significant association between the t(4;14) translocation and ID-1 expression in MM patients (P = 0.002). As high levels of ID-1 are associated with cancer, our findings indicate that MMSET promotes oncogenic transformation in t(4;14)+ MM patients by transcriptional activation of ID-1 expression.

α-1-Acid glycoprotein (AGP) is an acute-phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil-derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma-derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/enhancer-binding protein (C/EBP)-ɛ in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.

The present study compared the contents of pluripotent and lineage-committed hematopoietic progenitor cells (HPCs) in various types of allografts. Bone marrow (BM) allografts and single leukapheresis products (LPs) collected from G-CSF-mobilized donors contained similar amounts of pluripotent HPCs (CD34(+)CD38(-)) and total CD34(+) cells. However, the content of late-myeloid HPCs (CD34(+)CD33(+)CD15(+)) were significantly higher in BM grafts compared to LPs (P>0.02), whereas the contents of early-myeloid HPCs (CD34(+)CD33(+)CD15-) were 2.5-fold higher in LPs (P

This study investigated the expression pattern in primary plasma cells (PCs) of putative oncogenes suggested to be involved in multiple myeloma (MM) development. cDNA archives were generated by global reverse transcription polymerase chain reaction from CD38++/CD19-/CD56-/++ aberrant PCs of a prospective cohort of 96 subjects, including healthy individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), MM and MM with extramedullary manifestations (ExMM). The cDNA archives were analysed quantitatively for expression of the cyclin D1, fibroblast growth factor receptor 3 (FGFR3), C-MYC, C-MAF and cyclin D3 oncogenes. In addition, all patients were screened for IGH-MMSET hybrid transcripts. None of the analysed oncogenes was randomly distributed. C-MYC and cyclin D3 expression increased at the extramedullary transformation stage. Furthermore, C-MYC and cyclin D3 expression in CD56+ MM was similar to MGUS, whereas CD56- MM was similar to ExMM. FGFR3/IGH-MMSET was only observed among CD56+ MM patients, whereas an increased frequency of C-MAF dysregulation was seen among CD56- MM. High cyclin D1 expression levels were identified at similar frequencies at all stages, whereas the frequency of patients with low cyclin D1 levels increased during MM development. These data support the stepwise transformation model accumulating genetic alterations and proliferative capacity during MM initiation and development resulting in different clinical entities.

The pattern of expressed genes defines the structure and functional status of cells. Currently, most methods used in gene expression studies depend on large numbers of cells. Thus, their application may be hampered by the heterogeneity of cell populations, and by the low numbers of cells obtainable from in vivo sources. Such drawbacks may be overcome by methods suitable for the profiling of gene expression at the single cell level. We studied whether polymerase chain reaction (PCR) products synthesized from individual cells by global amplification of messenger RNA (mRNA) were suitable as probes for gene expression analysis. For this purpose, cells were subjected to reverse transcription and PCR using sequence independent primers (SIP RT-PCR). The resultant cDNA products were radiolabeled and hybridized to cDNA clones arrayed on a nylon membrane by vacuum slot blotting (a method referred to as slot blot analysis). The SIP RT-PCR procedure was reproducible and allowed the detection of twofold changes in input RNA copies per cell (range: 80-10.000 copies of an in vitro transcribed poly(A)-tailed RNA/cell). Analysis of total RNA and amplified cDNA, obtained from neutrophil granulocytes and the promyelocytic HL-60 cell line, demonstrated comparable gene expression profiles as measured by Northern blot and slot blot analysis. Slot blot analysis of HL-60 cells indicated that individual cells from an apparently homogenous population have varying expression of specific transcripts, which all contribute to the mRNA phenotype of their population. Interestingly, the genes that were detected in some but not all individual HL-60 cells were those found to peak within 2 days of retinoic acid-induced granulocytic differentiation. This study demonstrates the potential of cDNA, synthesized from individual cells by global amplification of mRNA, as probes for cDNA arrays.

The function of the mature polymorphonuclear neutrophil is dependent on its granules, each with its characteristic content of proteins. The granule proteins are formed at different stages during maturation of neutrophils from myeloblasts to segmented cells. The regulation of granule protein expression is controlled by a number of transcription factors, many of which are also essential for commitment of multipotent hematopoietic stem cells to lineage-committed myeloid progenitor cells and for differentiation of these progenitor cells; among these, PU.1 and C/EBPalpha stand out as critical for all granule proteins whereas AML-1 is critical for primary granule protein expression and C/EBPepsilon for secondary and tertiary granule protein expression.

Gene expression profiling has been used extensively to characterize cancer, identify novel subtypes, and improve patient stratification. However, it has largely failed to identify transcriptional programs that differ between cancer and corresponding normal cells and has not been efficient in identifying expression changes fundamental to disease etiology. Here we present a method that facilitates the comparison of any cancer sample to its nearest normal cellular counterpart, using acute myeloid leukemia (AML) as a model. We first generated a gene expression-based landscape of the normal hematopoietic hierarchy, using expression profiles from normal stem/progenitor cells, and next mapped the AML patient samples to this landscape. This allowed us to identify the closest normal counterpart of individual AML samples and determine gene expression changes between cancer and normal. We find the cancer vs normal method (CvN method) to be superior to conventional methods in stratifying AML patients with aberrant karyotype and in identifying common aberrant transcriptional programs with potential importance for AML etiology. Moreover, the CvN method uncovered a novel poor-outcome subtype of normal-karyotype AML, which allowed for the generation of a highly prognostic survival signature. Collectively, our CvN method holds great potential as a tool for the analysis of gene expression profiles of cancer patients.

Cytokine-mobilized peripheral blood progenitor cells (PBPCs) have been used successfully for hematopoietic reconstitution following allogeneic transplantation. The ease of harvest, the faster engraftment and the high yield of CD34+ cells have made this source of hematopoietic progenitor cells (HPCs) an attractive alternative to bone marrow (BM). In the present study we compared the engraftment potential of conventional BM allografts and single leukapheresis products (LPs) collected from healthy donors following the administration of granulocyte colony-stimulating factor (G-CSF). For this, lineage-committed and primitive HPCs were assessed by flow cytometry and by colony- and cobblestone area-forming cell (CFC, CAFC) assays. Mean numbers of CD34+ cells in LPs (n = 11) were similar to that of BM grafts (n = 12) (278+/-57 vs 227+/-34 x 10(6) CD34+ cells). The frequencies of CFCs, week 5 CAFCs and week 8 CAFCs were 1.6-, 8.4- and 10.3-fold higher in the CD34+ compartment of mobilized blood than that of marrow, resulting in significantly higher yields of clonogenic HPCs in LPs when compared to BM grafts. We conclude that G-CSF preferentially mobilizes clonogenic progenitors capable of short- and, in particular, longterm reconstitution, and that the engraftment potential of single LPs is superior to that of BM allografts. Hence, the use of PBPCs may be favorable for protocols that include graft manipulations with expected cell loss (eg T cell depletion, CD34+ selection). PBPCs may also be advantageous for gene therapy trials due to their high numbers of potential target cells (eg CAFCs).

The HemaExplorer (http://servers.binf.ku.dk/hemaexplorer) is a curated database of processed mRNA Gene expression profiles (GEPs) that provides an easy display of gene expression in haematopoietic cells. HemaExplorer contains GEPs derived from mouse/human haematopoietic stem and progenitor cells as well as from more differentiated cell types. Moreover, data from distinct subtypes of human acute myeloid leukemia is included in the database allowing researchers to directly compare gene expression of leukemic cells with those of their closest normal counterpart. Normalization and batch correction lead to full integrity of the data in the database. The HemaExplorer has comprehensive visualization interface that can make it useful as a daily tool for biologists and cancer researchers to assess the expression patterns of genes encountered in research or literature. HemaExplorer is relevant for all research within the fields of leukemia, immunology, cell differentiation and the biology of the haematopoietic system.

To the editor: The HemaExplorer Web server allows for an easy display of mRNA expression profiles for query genes in murine and human hematopoietic stem and progenitor cells that represent consecutive developmental stages along the myeloid differentiation pathway. In addition, it provides the

Background: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBP alpha is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. Methodology/Principal Findings: Here, we use mouse genetics to investigate the significance of C/EBP alpha serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. Conclusions/Significance: Taken together, our data shows that the substitution of C/EBP alpha serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context. (Less)

Chromosomal translocations of transcription factors generating fusion proteins with aberrant transcriptional activity are common in acute leukemia. In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic-acid receptor alpha (PML-RARA) fusion protein, which emerges as a consequence of the t(15;17) translocation, acts as a transcriptional repressor that blocks neutrophil differentiation at the promyelocyte (PM) stage. In this study, we used publicly available microarray data sets and identified signatures of genes dysregulated in APL by comparison of gene expression profiles of APL cells and normal PMs representing the same stage of differentiation. We next subjected our identified APL signatures of dysregulated genes to a series of computational analyses leading to (i) the finding that APL cells show stem cell properties with respect to gene expression and transcriptional regulation, and (ii) the identification of candidate drugs and drug targets for therapeutic interventions. Significantly, our study provides a conceptual framework that can be applied to any subtype of AML and cancer in general to uncover novel information from published microarray data sets at low cost. In a broader perspective, our study provides strong evidence that genomic strategies might be used in a clinical setting to prospectively identify candidate drugs that subsequently are validated in vitro to define the most effective drug combination for individual cancer patients on a rational basis.