Regulation Of Hematopoietic Cell Differentiation Research Articles

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

AMPK Downregulates Histone Gene Expression Via Dissociation of a Chromatin-Associated Transcription Complex in Response to Metabolic Stress in Acute Lymphoblastic Leukemia

We and others have reported that activation of the AMP-activated protein kinase (AMPK) mediates cell death in ALL. AMPK has been reported to interact with chromatin-associated proteins to epigenetically regulate gene expression in response to environmental/energy/metabolic stress, however, little is known about AMPK's epigenetic targets. To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we performed ChIP-seq with AMPKα2 and RNA pol II antibodies in Bp-ALL (KASUMI-2) and T-ALL (KE-37) cells treated with or without glucose or AICAR, a well known AMPK activator. ChIP-seq identified 132 and 167 significant AMPKα2 peaks located at gene promoters in AICAR-treated KASUMI-2 and KE-37 cells, respectively. GO terms analysis in KASUMI-2 control cells showed that AMPKα2 target set was enriched for genes involved in regulation of hematopoietic cell differentiation, DNA metabolism, DNA topology, and histone H3-K4 methylation, whereas in AICAR-treated cells it included the histone H3-K4 methylation, protein localization, Notch signaling, and mRNA destabilization. In AICAR-treated cells, we found that AMPKα2 was recruited to regions that were enriched for TAF1 and MYC targets, and to the promoter of the epigenetic regulators KMT2A and SETD1A. Among AMPKα2 targets, we identified a cluster of histone genes in which recruitment of AMPKα2 was significantly decreased at gene promoters. ChIP-seq data were validated by ChIP-qPCR on histone genes H1-2, H1-3, and H4C4, and the occupancy of AMPKα2 on histone gene loci in other Bp- and T-ALL cells confirmed. To correlate the level of gene mRNA expression and recruitment of AMPKα2 to chromatin we performed RNA-seq in KASUMI-2 and KE-37 cells treated ± AICAR for 24h. RNA-seq identified 1342 differential expressed genes in at least one of the cell lines, of which 57 were histone genes and 1285 non-histone genes. RT-qPCR confirmed histone gene mRNA downregulation in AICAR-treated cells. In addition, consistent with an epigenetic role for AMPK in ALL cells in response to energy/metabolic stress, we found that expression of the H3-K4 histone methyltransferase KMT2A and SETD1A genes were upregulated in KASUMI-2 cells treated with AICAR. To further investigate the effect of AMPK on histone gene expression, we generated HEK293 cells expressing single knockout (KO) or double knockout (DKO) of AMPKα1 and AMPKα2 genes using CRISPR/Cas9, and determined the level of selected histone gene mRNA expression using RT-qPCR. Our data show that both AMPKα1 and AMPKα2 participate in regulating histone gene expression, and these findings were confirmed using MEF WT, AMPKα1/KO, AMPKα2/KO, and AMPKα1/α2/DKO cells. These data support our previous findings indicating that AMPK kinase activity was required for histone genes’ regulation in response to metabolic stress. Next, we examined the cluster of non-histone genes altered by AICAR, and found that downregulated genes were enriched in MYC, Myc-associated factor X, and transcription initiation factor TFIID subunit 1 (TAF1) targets, and involved in pathways such as gene expression and protein metabolism. In contrast, the upregulated genes were enriched in RUNX1, SPI1, and GATA1 targets, and involved in pathways related to immune system signaling, regulation by TP53, and apoptosis. Using HEK293 CRISPR AMPKα1/KO, AMPKα2/KO and AMPKα1/α2/DKO cells, we demonstrated that AMPK is also required for MYC expression. In conclusion, our data show that (i) in response to metabolic stress, AMPKα2 is recruited to or released from a chromatin-associated complex to regulate gene transcription in ALL, (ii) AMPKα2 targets include the histone, MYC-target, and the epigenetic regulator KMT2A and SETD1A genes (H3-K4 histone methyltransferases), and (iii) AICAR treatment represses histone gene transcription via an AMPK-dependent mechanism. Further elucidation of AMPK's interactions with members of a putative AMPK/chromatin-associated transcription complex may lead to unique opportunities for epigenetic-based therapeutic interventions to exploit synthetic lethality in relapse/refractory ALL and other hematological malignancies.

Insights Into the Roles of GATA Factors in Mammalian Testis Development and the Control of Fetal Testis Gene Expression.

Defining how genes get turned on and off in a correct spatiotemporal manner is integral to our understanding of the development, differentiation, and function of different cell types in both health and disease. Testis development and subsequent male sex differentiation of the XY fetus are well-orchestrated processes that require an intricate network of cell-cell communication and hormonal signals that must be properly interpreted at the genomic level. Transcription factors are at the forefront for translating these signals into a coordinated genomic response. The GATA family of transcriptional regulators were first described as essential regulators of hematopoietic cell differentiation and heart morphogenesis but are now known to impact the development and function of a multitude of tissues and cell types. The mammalian testis is no exception where GATA factors play essential roles in directing the expression of genes crucial not only for testis differentiation but also testis function in the developing male fetus and later in adulthood. This minireview provides an overview of the current state of knowledge of GATA factors in the male gonad with a particular emphasis on their mechanisms of action in the control of testis development, gene expression in the fetal testis, testicular disease, and XY sex differentiation in humans.

The Role of NKL Homeobox Genes in T-Cell Malignancies

Homeobox genes encode transcription factors controlling basic developmental processes. The homeodomain is encoded by the homeobox and mediates sequence-specific DNA binding and interaction with cofactors, thus operating as a basic regulatory platform. Similarities in their homeobox sequences serve to arrange these genes in classes and subclasses, including NKL homeobox genes. In accordance with their normal functions, deregulated homeobox genes contribute to carcinogenesis along with hematopoietic malignancies. We have recently described the physiological expression of eleven NKL homeobox genes in the course of hematopoiesis and termed this gene expression pattern NKL-code. Due to the developmental impact of NKL homeobox genes these data suggest a key role for their activity in the normal regulation of hematopoietic cell differentiation including T-cells. On the other hand, aberrant overexpression of NKL-code members or ectopical activation of non-code members has been frequently reported in lymphoid and myeloid leukemia/lymphoma, demonstrating their oncogenic impact in the hematopoietic compartment. Here, we provide an overview of the NKL-code in normal hematopoiesis and discuss the oncogenic role of deregulated NKL homeobox genes in T-cell malignancies.

Structural Changes in Transcriptional Regulatory Networks for Cell-type-specific Gene Expression During Hematopoiesis

Hematopoiesis is an extensively studied model system for cell differentiation. Cell-type-specific gene expression patterns are observed during hematopoiesis. Gene expression is governed by regulatory networks composed of cell-type-specific transcription factors. Resolving the transcriptional regulatory network for cell-type-specific gene expression provides a promising means of understanding the mechanisms underlying cell fate decisions. In this study, transcriptional regulatory networks in hematopoietic stem and progenitor cells were predicted based on gene expression profiles and distributions of transcription factor binding motifs in the promoter regions of cell-type-specific transcription factors. In particular, structural changes that occur when pluripotent stem cells progress to lineage-committed progenitors were evaluated. Marked changes in the regulatory circuit of transcription throughout the differentiation process could be elucidated by network analysis. Modular structures were a frequently described feature of biological networks observed in estimated networks. Within a module, most transcription factors were found to be regulated by a small number of regulators acting as downstream targets. Certain regulators within these modules coincide with known key regulators of hematopoietic cell differentiation. In addition to the modular structure, a twolayered structure was clearly observed in progenitor regulatory networks. Transcription factors could be distinctly divided into regulators within the regulatory layer and into targets in the output layer according to their degree of distribution. The restriction of mutual regulation between transcription factors was remarkable in that it allowed for alterations in network structures between hematopoietic stem cells and progenitors. Thus, using this approach, the relationships among transcription factors could be revealed by a reduction in mutual regulation to form a modular structure within the regulatory network

The key roles of the lysine acetyltransferases KAT6A and KAT6B in physiology and pathology.

Histone modifications and more specifically ε-lysine acylations are key epigenetic regulators that control chromatin structure and gene transcription, thereby impacting on various important cellular processes and phenotypes. Furthermore, lysine acetylation of many non-histone proteins is involved in key cellular processes including transcription, DNA damage repair, metabolism, cellular proliferation, mitosis, signal transduction, protein folding, and autophagy. Acetylation affects protein functions through multiple mechanisms including regulation of protein stability, enzymatic activity, subcellular localization, crosstalk with other post-translational modifications as well as regulation of protein-protein and protein-DNA interactions. The paralogous lysine acetyltransferases KAT6A and KAT6B which belong to the MYST family of acetyltransferases, were first discovered approximately 25 years ago. KAT6 acetyltransferases acylate both histone H3 and non-histone proteins. In this respect, KAT6 acetyltransferases play key roles in regulation of transcription, various developmental processes, maintenance of hematopoietic and neural stem cells, regulation of hematopoietic cell differentiation, cell cycle progression as well as mitosis. In the current review, we discuss the physiological functions of the acetyltransferases KAT6A and KAT6B as well as their functions under pathological conditions of aberrant expression, leading to several developmental syndromes and cancer. Importantly, both upregulation and downregulation of KAT6 proteins was shown to play a role in cancer formation, progression, and therapy resistance, suggesting that they can act as oncogenes or tumor suppressors. We also describe reciprocal regulation of expression between KAT6 proteins and several microRNAs as well as their involvement in cancer formation, progression and resistance to therapy.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling.

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Noncoding RNAs in Cardiovascular Disease: Pathological Relevance and Emerging Role as Biomarkers and Therapeutics.

Noncoding RNAs (ncRNA) include a diverse range of functional RNA species-microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) being most studied in pathophysiology. Cardiovascular morbidity is associated with differential expression of myriad miRNAs; miR-21, miR-155, miR-126, miR-146a/b, miR-143/145, miR-223, and miR-221 are the top 9 most reported miRNAs in hypertension and atherosclerotic disease. A single miRNA may have hundreds of messenger RNA targets, which makes a full appreciation of the physiologic ramifications of such broad-ranging effects a challenge. miR-21 is the most prominent ncRNA associated with hypertension and atherosclerotic disease due to its role as a "mechano-miR", responding to arterial shear stresses. "Immuno-miRs", such as miR-155 and miR-223, affect cardiovascular disease (CVD) via regulation of hematopoietic cell differentiation, chemotaxis, and activation in response to many pro-atherogenic stimuli. "Myo-miRs", such as miR-1 and miR-133, affect cardiac muscle plasticity and remodeling in response to mechanical overload. This in-depth review analyzes observational and experimental reports of ncRNAs in CVD, including future applications of ncRNA-based strategies in diagnosis, prediction (e.g., survival and response to small molecule therapy), and biologic therapy.

MicroRNA-486 regulates normal erythropoiesis and enhances growth and modulates drug response in CML progenitors

AbstractMicroRNAs (miRNAs) are key regulators of hematopoietic cell differentiation and may contribute to altered growth of leukemic stem cells. Using microarray-based miRNA profiling, we found that miRNA 486 (miR-486) is significantly upregulated in chronic myeloid leukemia (CML) compared with normal CD34+ cells, particularly in the megakaryocyte-erythroid progenitor population. miR-486-5p expression increased during erythroid differentiation of both CML and normal CD34+ cells. Ectopic miR-486-5p expression enhanced in vitro erythroid differentiation of normal CD34+ cells, whereas miR-486-5p inhibition suppressed normal CD34+ cell growth in vitro and in vivo and inhibited erythroid differentiation and erythroid cell survival. The effects of miR-486-5p on hematopoietic cell growth and survival are mediated at least in part via regulation of AKT signaling and FOXO1 expression. Using gene expression and bionformatics analysis, together with functional screening, we identified several novel miR-486-5p target genes that may modulate erythroid differentiation. We further show that increased miR-486-5p expression in CML progenitors is related to both kinase-dependent and kinase-independent mechanisms. Inhibition of miR-486-5p reduced CML progenitor growth and enhanced apoptosis following imatinib treatment. In conclusion, our studies reveal a novel role for miR-486-5p in regulating normal hematopoiesis and of BCR-ABL–induced miR-486-5p overexpression in modulating CML progenitor growth, survival, and drug sensitivity.

Down-Regulation of Homeobox Gene Ventx Promotes Expansion of Human Bone Marrow Hematopoietic Stem Cells (HSC)

Abstract 1272Hematopoietic stem cells (HSC) give rise to mature cells of all lineages of blood and immune systems. HSC transplantation has shown great promise in the treatment of malignancies, reconstitution of hematopoietic systems and HSC-based gene therapy. Cell intrinsic factors/pathways have been the targets of intensive investigation for its potential application in HSC expansion. Over the past decades, several critical cell fate determination pathways, such as the Wnt signaling pathways and senescence pathways have been implicated in the proliferation and differentiation of HSC. Moreover, overexpression of HoxB4 and BMI1 was found to be able to expand human HSC 2∼3 folds. Nevertheless, the regulatory mechanisms of HSC proliferation and differentiation remain incompletely understood and safe and efficacious expansion of human HSC remains as a fundamental challenge that limits the clinical application of HSC-based therapy.VentX is a human homologue of the Xenopus homeobox protein Xom of the BMP4 signaling pathway. Using Xenopus model and methods of reverse genetics, our recent work showed that VentX is a LEF/TCF associated Wnt repressor and an activator of senescence pathways. VentX expression is highly regulated and restricted in hematopoietic cells and serves a major regulator of hematopoietic cell differentiation. To explore the potential role of VentX in proliferation and differentiation of HSC during hematopoiesis, we quantified VentX expression during hematopoiesis, using qRT-PCR methods and examined the effects of altered VentX expression on HSC properties in vitro and in vivo.Our data showed that VentX expression is significantly up-regulated during oncogenesis of hematopioetic cells. We demonstrated that lentiviral knockdown of VentX allowed for more than 5 fold ex vivo expansion of human HSC with balanced lineage development. Importantly, transient knockdown of VentX by siRNA also led to expansion of HSC. The effect of VentX down-regulation on the expansion of human HSC was also demonstrated by enhanced engraftment in the SCID/NODγ2null mouse model. Consistent with its role as a novel regulator of HSC, overexpression of VentX significantly inhibited clonal genesis of HSC. Mechanistically, we demonstrated that VentX controls the expression of cell cycle regulators downstream of the Wnt and senescence pathways, such as the C-myc, CyclinD1 and p21.In summary, using methods of reverse genetic and developmental modeling, we identified VentX as a novel regulator for expansion of human BM HSC. The results of our investigations provide novel insight in regulating HSC proliferation and differentiation. In addition, the findings that transient down-regulation of VentX by SiRNA lead to efficient expansion of bone marrow HSC suggests that VentX may serve as a novel target for safe expansion of HSC for its potential clinical applications. Disclosures:No relevant conflicts of interest to declare.

PU.1 Is a Downstream Target of C/EBPα in Normal Hematopoiesis and Acute Myeloid Leukemia

Abstract 1353The transcription factors PU.1 and C/EBPα are key regulators of hematopoietic cell differentiation. Tight and coordinated regulation of these factors is essential for normal hematopoiesis and even moderate alterations can lead to acute myeloid leukemia (AML). Previous studies established that in PU.1 knockout mice myeloid differentiation is blocked at an earlier stage compared to C/EBPα knockouts, consistent with PU.1 acting upstream of C/EBPα during hematopoietic differentiation. Recently, however, we and others identified a PU.1 upstream regulatory element (URE) which contains potential C/EBP binding sites. C/EBPα binds to the PU.1 URE in vitro and in vivo. Furthermore, C/EBPα transactivated the PU.1 proximal promoter in a URE dependent manner. We, therefore, hypothesized that PU.1 is a target gene of C/EBPα in hematopoietic cells.To assess the role of PU.1 as a downstream target of C/EBPα in normal hematopoiesis we performed gene expression analysis in immature hematopoietic cells of conditional C/EBPα knockout mice (Mx1-Cre). Of note, we observed a strong reduction of PU.1 expression in hematopoietic stem cells (HSCs: CD150+CD48-LSK) after excision of C/EBPα, corroborating that PU.1 is a target of C/EBPα in murine HSCs in vivo. Moreover, lentiviral PU.1 expression alleviated the myeloid differentiation block of C/EBPα−/− KSL cells as evidenced by the differentiation to Gr-1 and Mac1 positive myeloid cells.Targeted deletion of the PU.1 URE reduces PU.1 expression and induces myeloid leukemia. Additionally, inactivation of C/EBPα by various mechanisms is a common observation in many AML subtypes. Therefore, we tested if dysregulation of C/EBPα is associated with decreased PU.1 expression. Gene expression studies in several human AML cell lines revealed a positive correlation between C/EBPα and PU.1 expression. Furthermore, we analyzed expression of C/EBPα and PU.1 in a well characterized cohort of 285 AML patients. Importantly, PU.1 expression was strongly reduced in cases with either C/EBPα mutations or C/EBPα promoter silencing compared to other AML subtypes.Taken together, our data support that PU.1 is a downstream target gene of C/EBPα in normal hematopoiesis as well as human leukemia. We currently develop a mouse model containing targeted mutations of three C/EBP binding sites in the PU.1 URE. This model will help to further pinpoint the functional impact of C/EBPα mediated regulation of PU.1 in different hematopoietic populations and to determine how this regulation may contribute to leukemia development in vivo.The first two authors contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

Human adenylate kinase 2 deficiency causes a profound hematopoietic defect associated with sensorineural deafness

Reticular dysgenesis is an autosomal recessive form of human severe combined immunodeficiency characterized by an early differentiation arrest in the myeloid lineage and impaired lymphoid maturation. In addition, affected newborns have bilateral sensorineural deafness. Here we identify biallelic mutations in AK2 (adenylate kinase 2) in seven individuals affected with reticular dysgenesis. These mutations result in absent or strongly decreased protein expression. We then demonstrate that restoration of AK2 expression in the bone marrow cells of individuals with reticular dysgenesis overcomes the neutrophil differentiation arrest, underlining its specific requirement in the development of a restricted set of hematopoietic lineages. Last, we establish that AK2 is specifically expressed in the stria vascularis region of the inner ear, which provides an explanation of the sensorineural deafness in these individuals. These results identify a previously unknown mechanism involved in regulation of hematopoietic cell differentiation and in one of the most severe human immunodeficiency syndromes.

Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation

Inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs play an important regulatory role in immune cell activation. In addition, several studies suggest that these receptors are involved in the regulation of hematopoietic cell differentiation. Here, we have investigated the expression of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), an inhibitory receptor expressed on most peripheral blood leukocytes and on CD34+ hematopoietic progenitor cells, in neutrophil differentiation and activation. We found that although LAIR-1 was expressed on peripheral blood eosinophils, cell-surface expression on mature neutrophils was low, suggesting that LAIR-1 expression is regulated during granulocyte differentiation. Indeed, the promyeloid cell line HL-60 expressed LAIR-1, but the expression decreased during chemical-induced differentiation toward neutrophils. Similarly, in bone marrow-derived neutrophil precursors, the most immature cells expressed LAIR-1, and loss of LAIR-1 expression was associated with neutrophil maturation. LAIR-1 was re-expressed rapidly on the membrane of mature neutrophils upon stimulation with tumor necrosis factor alpha, granulocyte macrophage-colony stimulating factor, or N-formyl-methionyl-leucyl-phenylalanine, indicating that LAIR-1 may also regulate neutrophil effector function. Our studies suggest that LAIR-1 may play a regulatory role in differentiation and function of human granulocytes.

Ikaros increases normal apoptosis in adult erythroid cells

Ikaros is a critical transcriptional regulator of hematopoietic cell differentiation. In addition to its effects on the lymphoid system and hematopoietic stem-cell compartment, we have previously shown that Ikaros is also required for normal erythroid development. In this report, we compare Ikaros-dependent gene expression in erythroid cells of mice lacking the Ikaros protein with that of normal mice in purified adult bone-marrow erythroid cells (BMRC). Gene expression, measured by Affymetrix microarray analysis, indicates that in the BMRC of Ikaros-null mice, there is significant up-regulation of SMADs 6 and 7, serine protease inhibitor 3, and immediate-early protein 3 (IER3), all proteins that play a modulating role in apoptosis. We investigate the role of Ikaros in oxidative stress-induced apoptosis using Annexin-V staining and FACS analysis. We find a decrease in apoptosis in the BMRC of Ikaros-null mice compared to normal mice. This effect is also seen in nonerythroid cells but is stronger in BMRC. We conclude that normal Ikaros function increases normal apoptosis in erythroid cells. The data also suggest that Ikaros plays a role in apoptosis-mediated events in other normal hematopoietic cell lineages.

Differentiation primary response genes and proto-oncogenes as positive and negative regulators of terminal hematopoietic cell differentiation.

By genetically manipulating hematopoietic cells of the myeloid lineage, including both normal cells and differentiation inducible leukemic cell lines, evidence was obtained to indicate that myeloid differentiation primary response (MyD) genes and proto-oncogenes, which are known to control cell growth, function as positive and negative regulators of terminal hematopoietic cell differentiation, which is associated with inhibition of cell growth, and, ultimately programmed cell death (apoptosis). Interferon regulatory factor-1 (IRF-1), an MyD gene induced by Interleukin 6 (IL-6) or Leukemia Inhibitory factor (LIF), plays a role in growth inhibition associated with terminal differentiation. Leucine zipper transcription factors of the fos/jun family, also identified as MyD genes, function as positive regulators of hematopoietic cell differentiation, increasing the propensity of myeloblastic leukemia cells to be induced for differentiation in vitro, and reducing the aggressiveness of their leukemic phenotype in vivo. The zinc finger transcription factor EGR-1, an MyD gene specifically induced upon macrophage differentiation, was shown to be essential for and to restrict differentiation along the macrophage lineage. Finally, evidence has been accumulating to indicate that the novel MyD genes--MyD116, MyD118 and gadd45 (a member in the MyD118 gene family)--play a role in growth arrest and apoptosis of hematopoietic cells, as well as other cell types. The proto-oncogenes c-myc and c-myb, known to regulate cellular growth, were shown to function as negative regulators of terminal differentiation. Both c-myc and c-myb are normally expressed in proliferating myeloblasts and suppressed following induction of differentiation. Deregulated and continuous expression of c-myc was shown to block terminal myeloid differentiation at an intermediate stage in the progression from immature blasts to mature macrophages, whereas deregulated and continuous expression of c-myb blocked the terminal differentiation program at the immature myeloblast stage. By manipulating myc function in conditional (differentiation inducible) mutant myeloblastic leukemia cell lines, expressing a chimeric mycer transgene, it was shown that there is a window during myeloid differentiation, after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, and where activation of c-myc has no apparent effect on mature macrophages. These myeloblastic leukemia cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal hematopoiesis and in leukemogenesis, while also providing a strategy to clone myc target genes.(ABSTRACT TRUNCATED AT 250 WORDS)

The stimulation of rat bone marrow fibroblast colony formation by 2-mercaptoethanol.

The definition of the function of bone marrow stromal cells in the regulation of hematopoiesis has been complicated by the limited growth of these cells in vitro. We have demonstrated that the addition of 2-mercaptoethanol to rat bone marrow cultures enhances bone marrow fibroblast proliferation as evidenced by a 2-fold increase in the total number of fibroblast colonies and a 5-fold increase in the number of these colonies which contain more than 80 cells. We also present evidence suggesting that enhancement of fibroblast growth may not be due to direct action of 2-mercaptoethanol, but may result from the activation of a serum component. The results from this study should facilitate further research into the function of bone marrow fibroblasts in the regulation of hematopoietic cell differentiation.