Proliferation Of Myeloid Progenitors Research Articles

Epigenetic Signaling Is Required for HoxA9-Based Leukemic Transformation.

Abstract 3966Poster Board III-902Since oncogenic activation of HoxA9 is induced by multiple chromosomal translocations affecting MLL1 (11q23)(e.g. MLL-Af9) or Nup98 (11p15)(e.g. Nup98-HoxA9 or Nup98-Nsd1), the function of the HoxA9 transcription factor is of critical interest in human acute myeloid leukemia (AML). HoxA9 forms heterodimeric DNA binding complexes with members of the Pbx and/or Meis family of homeodomain proteins. Importantly, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. In a transcriptional circuit conserved to Drosophila, we have recently shown that Gfi1 represses HoxA9, Meis1 and Pbx1 expression, that Gfi1 and HoxA9 demonstrate dramatic epistatic relationships, and that Gfi1 loss of function is potently preleukemic. Our new bioinformatic, biochemical and expression data reveal microRNA genes to be targets of endogenous HoxA9 versus Gfi1 antagonism. Moreover, these miR are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts oncogenic signaling by HoxA9, Nup98-HoxA9 and MLL-Af9 (but not AML-ETO which does not signal through HoxA9). In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia lethality. These data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins. Disclosures:No relevant conflicts of interest to declare.

PLZF is a regulator of homeostatic and cytokine-induced myeloid development

A major question in hematopoiesis is how the system maintains long-term homeostasis whereby the generation of large numbers of differentiated cells is balanced with the requirement for maintenance of progenitor pools, while remaining sufficiently flexible to respond to periods of perturbed cellular output during infection or stress. We focused on the development of the myeloid lineage and present evidence that promyelocytic leukemia zinc finger (PLZF) provides a novel function that is critical for both normal and stress-induced myelopoiesis. During homeostasis, PLZF restricts proliferation and differentiation of human cord blood-derived myeloid progenitors to maintain a balance between the progenitor and mature cell compartments. Analysis of PLZF promoter-binding sites revealed that it represses transcription factors involved in normal myeloid differentiation, including GFI-1, C/EBPalpha, and LEF-1, and induces negative regulators DUSP6 and ID2. Loss of ID2 relieves PLZF-mediated repression of differentiation identifying it as a functional target of PLZF in myelopoiesis. Furthermore, induction of ERK1/2 by myeloid cytokines, reflective of a stress response, leads to nuclear export and inactivation of PLZF, which augments mature cell production. Thus, negative regulators of differentiation can serve to maintain developmental systems in a primed state, so that their inactivation by extrinsic signals can induce proliferation and differentiation to rapidly satisfy increased demand for mature cells.

Exclusion of ABCB8 and ABCB10 as cancer candidate genes in acute myeloid leukemia

Acute myeloid leukemia (AML) exhibits great heterogeneity in manifestation, sensitivity to therapy and genetic basis of pathology. Numerous studies have implied that AML arises from the sequential accumulation of mutations at the level of the hematopoietic stem/progenitor cell, resulting in a disturbed balance of proliferation, differentiation and apoptosis of immature myeloid progenitors.1 To gain more insight into somatic mutations that affect biological pathways in tumorigenesis, a systematic mutation analysis of virtually all annotated human protein-coding genes in eleven breast and eleven colorectal cancers has been performed,2 leading to the identification of 280 genes as candidate cancer (CAN) genes. Importantly, several of the identified CAN genes are known to be mutated in hematological malignancies, including CBFA2T3 (ETO2), FBXW7, KRAS, NF1, PTEN, RUNX1T1 and TP53.1, 2 Several genes newly identified to be mutated in breast and colorectal cancers belong to the class of ATP-binding cassette (ABC) transporters,2 which are known to affect the response to therapy of AML and other cancers by conferring chemoresistance. Two of the ABC transporters discovered to harbor novel somatic mutations in breast cancer, the mitochondrial transporters ABCB8 and ABCB10, are highly expressed in both normal and leukemic hematopoietic stem cells.3 ABCB8 and ABCB10 are structurally closely related and are evolutionary highly conserved, suggesting a fundamental function in cellular physiology. Although their functions have not been completely elucidated, some clues regarding their biological roles can be postulated on the basis of homologues of ABCB10 in other species. Firstly, multi-drug resistance like 1 (MDL1), the ABCB10 homologue from Saccharomyces cerevisiae, regulates cellular responses to oxidative stress, such as reactive oxygen species (ROS).4 Reactive Oxygen Species signals stimulate proliferation and induce genetic instability in malignant transformed cells, conferring increased basal oxidative stress in cancer cells.5 ABCB10 might represent an important factor in mediating the response to oxidative stress in cancer cells. Secondly, the murine ABCB10 homologue, ABC-mitochondria erythroid (ABC-me), is a direct target of the transcription factor GATA1 and overexpression of murine ABCB10 enhances hemoglobin synthesis.6 Furthermore, a recent study identified ABCB10 to be essential for proliferation of human mammary epithelial cells.7 Thus, mutations in ABCB10 may cause resistance to oxidative stress, thereby contributing to cancer development. In this context, we pursued to investigate whether ABCB8 and ABCB10 are CAN genes in AML. To this end, the coding regions of ABCB8 and ABCB10 were screened by heteroduplex analysis (HA) using genomic DNA isolated from diagnostic bone marrow or peripheral blood samples from a panel of 94 AML patients who were referred to the Department of Hematology at the Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, between 1991 and 2004. To identify potential mutations that affect splicing, the first 15 intronic nucleotides surrounding the intron–exon junctions were analyzed as well. Amplicons covering relevant regions were generated by a two-step polymerase chain reaction (PCR) followed by HA (for details see Supplementary Methods and Supplementary Figure S1 and 2; for primer sequences see Supplementary Table S1). Fragments exhibiting altered peak patterns in HA were subsequently sequenced to identify the nucleotide changes. Exons with multiple known single nucleotide polymorphisms (SNPs) were subjected to direct sequencing. A limitation of the HA method is that homozygous mutations or SNPs will not be detected. As all reported somatic mutations in ABCB8 and ABCB10 are heterogeneous,2 we reasoned that most point mutations in the cohort would be detected by HA.

CSF3R mutations in paediatric acute myeloid leukaemia

<i>CSF3R</i> mutations in paediatric acute myeloid leukaemia

Hedgehog Signaling Regulates HSC Proliferation.

Hedgehog (Hh) signaling is essential for normal development and is dysregulated in many cancers. Hh signaling is active in normal bone marrow and the majority of acute myeloid leukemias, however, the precise role of Hh signaling and its positive effector Gli1 in normal or malignant hematopoiesis is not known. We have analyzed the bone marrow of Gli1 null mice to understand the role of this transcription factor in normal hematopoiesis in order to gain insight into its potential role in leukemia. Gli1 null mice develop normally and have normal peripheral blood counts but the bone marrow shows skewing of the c-Kit+Sca1+Lin-neg (KSL) progenitor compartment with increased CD34negKSL long-term HSC (LT-HSC) and decreased 34+KSL short-term HSC (ST-HSC). An analogous difference was observed in the c-Kit+Sca1negLinneg (KL) myeloid progenitor compartment with an increase in FcRγlowCD34+KL common myeloid progenitors (CMP) and decrease in the FcRγhighCD34+KL granulocyte monocyte progenitors (GMP). We speculated that these differences could be due to impaired cell cycle since both the ST-HSC and GMP are more proliferative than LT-HSC and CMP, respectively. Cell cycle analysis by DNA content and BrdU pulse labeling (100mg/kg IP 14 hours prior to analysis) revealed a marked decrease of proliferation in the LT-HSC, ST-HSC, CMP, and GMP compartments of Gli1 null mice. We supported this conclusion by demonstrating that the bone marrow of Gli1 null mice are relatively radio-resistant. Mice exposed to 400 cGy of total body irradiation followed with serial blood counts revealed less severe nadir, but delayed rebound of white blood cells in Gli1 null mice. We further hypothesized that although Gli1 appears to be dispensable for steady-state peripheral hematopoiesis, it might be necessary for rapid proliferation of progenitors needed during stressed hematopoiesis. In brain development, where Hh signaling is much better understood, active Hh signaling is critical for regulating proliferation of neural stem cells and Gli1 activity significantly increases after depletion of neural progenitors with chemotherapy (Bai et al., Development, 2002). To extend this observation to hematopoiesis, we treated Gli1 null mice and wild-type litter-mates with 5-fluorouracil (5-FU) at 100mg/kg and measured serial blood counts. Gli1 null mice had a delayed recovery of total white blood cells and neutrophil counts at 6 days after 5-FU, but this difference normalized by 20 days after treatment. To confirm that this difference was due to impaired proliferation and not increased sensitivity to 5-FU, we treated Gli1 null and wild-type mice with G-CSF (10mcg/kg/day) for three days to stimulate neutrophil proliferation. Confirming our hypothesis, we observed an attenuated neutrophil response in G-CSF stimulated Gli1 null mice. In summary, we have demonstrated that Gli1 loss leads to decreased HSC and myeloid progenitor proliferation, which has important functional consequences for stress hematopoiesis. These data suggest that abnormal Hh activity in leukemia may be important for driving the uncontrolled proliferation of cancer cells. Gli1 null mice were a kind gift from Alexandra Joyner, Memorial Sloan-Kettering Cancer Center

Understanding the Role of TRKA Receptor in Acute Myeloid Leukemias: From Proliferation and Pro-Survival Signals to a Novel Therapeutic Approach

Background: TRKA (Tropomyosin Receptor Kinase A) is a member of the neurotrophin tyrosine kinase receptor family. Nerve Growth Factor (NGF) binds specifically to TRKA triggering multiple downstream pathways which regulate proliferation and differentiation in a number of neural, epithelial and epidermal cell types. TRKA is also expressed in hematopoietic progenitor cells and NGF has been demonstrated to promote the colony growth and differentiation of myeloid progenitor cells. Interestingly, Pralle et al. found that 44% of patients with acute myeloid leukemia (AML) express TRKA mRNA and up to 30% encode activating TRKA mutations. Furthermore, a constitutively-active TRKA variant has been shown to contribute to myeloid leukemogenesis.Purpose: To evaluate the role of NGF/TRKA signaling on AML proliferation and survival through effects on downstream signaling pathways, and evaluate the effects of TRK inhibition on human AML.Methods: TRKA protein expression was measured in a panel of human AML cell lines (U937, K562, THP1, NB4, HL60, KU812). AlamarBlue (MTT) assays were used to measure proliferation/viability. For proliferation assays, cells were stimulated with NGF (5 ng/ml) for 1–3 days with or without the addition of a TRK inhibitor AZ-23 (25nM). Cell counts, cell cycle and survival were measured using trypan blue cell counts, and propidium iodine-based flow cytometry assays. Downstream signaling events were measured following a 15 minute exposure to recombinant human NGF. Antibodies for phosphorylated and total AKT, ERK, JNK and MEKK were used. In a human AML xenograft model, mice were treated with 100 mg/kg (2.5 mg) of AZ-23 by gavage for 2 weeks without toxicity. Weekly tail vein blood collection and flow cytometry were used to evaluate leukemia tumor burden. Kaplan-Meier curves were used to assess effects on morbidity.Results: Five of six human AML cell lines showed detectable TRKA protein levels by Western. When stimulated with NGF, all five cell lines which expressed TRKA demonstrated enhanced proliferation within 24 hours with mean value of 20% (10 to 45%, p=<0.001), which was abrogated with 25nM AZ-23 (p<0.01). The HL60 cell line showed the most impressive response to NGF (fig 1). The AML cell line which did not express TRKA (U937) and a human pre-B ALL cell line (Nalm-6) did not respond to NGF stimulation, as expected. There were no significant difference between only AZ-23 and the control group (p=>0.5). Following starvation, NGF stimulation induced the phosphorylation of ERK and AKT (2–4 fold), while inhibiting the phosphorylation of MEKK and JNK. All of these signaling effects were blocked with addition of AZ-23. Importantly, human AML-engrafted mice treated with the TRKA inhibitor AZ-23 had a significant decreased AML burden at 3 weeks post-inoculation (70% vs. 18% p=0.004). Kaplan-Meier estimates showed a Hazard Ratio of 10.5 (p=0.01) fig 3.Conclusions: TRKA has been implicated in the proliferation and transformation of myeloid progenitors and may be expressed and activated in a significant subset of AML cases. Our studies provide evidence of an important role for NGF/TRKA signaling in human AML. We show that NGF induces TRK-dependent proliferation while inducing ERK and AKT phosphorylation, consistent with a proliferative and pro-survival effect. Furthermore, we show that NGF stimulation inhibits the phosphorylation of MEKK and JNK, which are associated with NGF withdrawal and promote apoptosis. Finally, we demonstrate in a human AML xenograft model that a selective TRK inhibitor AZ-23 can effectively reduce AML burden and enhance survival. Based on these results, we propose that TRK inhibition may be a therapeutic option for a subgroup of AML, and warrants further pre-clinical development. [Display omitted] [Display omitted]

Growth of p210Bcr-Abl Transformed Human Hematopoietic Stem Cells in Immunodeficient Mice.

Expression of the p210Bcr-Abl fusion gene in primary hematopoietic stem and progenitor cells by transduction with Bcr-Abl-encoding retroviruses is a useful approach to investigate CML pathogenesis. We have previously shown that human CD34+ cells expressing BCR/ABL demonstrate several features characteristic of primary CML progenitors including increased proliferation in committed and primitive progenitor culture, reduced adhesion to fibronectin, and reduced chemotaxis to stroma-derived factor-1alpha. Here we investigated the growth of Bcr-Abl transformed human hematopoietic stem cells (HSC) in immunodeficient NOD/SCID IL2Rγ chain KO (NSG) mice. Human cord blood (CB) CD34+ cells were transduced with retroviral vectors expressing the p210Bcr-Abl gene and transplanted via tail vein injection into sub-lethally irradiated (300 cGy) 8 weeks old NSG mice. Mice were euthanized at different times after transplant and human cell engraftment was analyzed by flow cytometry. We observed a progressive increase in human CD45+ve cells in bone marrow of injected mice over time (% human CD45+ve cells in BM: Wk 2, 28±5,n=3; Wk 4, 57±6,n=3; Wk 8, 79±3,n=6; Wk 12, 80±5,n=7; Wk16–20, 95±2,n=3; total number of human CD45+ cells in both femora in millions: Wk 2, 0.1±0, Wk 4, 2.1±0.5, Wk 8, 9.7±1.8, Wk 12, 6.5±2.4, Wk 16–20, 14.5±6.2). The number of GFP expressing cells also increased over time up to 20 weeks after transplantation (GFP+ cells in both femora in millions: Wk 2, 0.01±0, Wk 4, 0.38±0.08, Wk 8, 0.81±0.27, Wk 12, 0.91±0.39, Wk16–20, 2.58±2.27). We confirmed using quantitative RT-PCR and Western blot analysis that engrafted cells expressed the Bcr-Abl gene, and that Bcr-Abl expression was restricted to GFP+ cells. We further observed that mice started developing splenomegaly 12 weeks after transplantation (spleen weight in grams: Wk 8, 36±4, Wk12, 196±56, Wk16–20, 207±55; spleen cell counts in millions: Wk 8, 2.6±1.0, Wk 12, 11.2±4.2, Wk 16–20, 18.6±10.5). Analysis of the phenotype of engrafted GFP+ human cells indicated that both myeloid and lymphoid GFP+ cells were generated. The majority of engrafted cells expressed B-lymphoid markers, consistent with the usual engraftment pattern of CB CD34+ cells in this mouse model, (% CD19+ cells in BM at Wk 8, 75±6, Wk 16–20, 51±17, % CD33+ cells in BM at Wk 8, 34±12, Wk16–20, 27±14). Two mice demonstrated very high percentages of GFP expressing B-lymphoid cells, consistent with clonal outgrowth of Bcr-Abl expressing B-cell progenitor population. To confirm that Bcr-Abl-expression was achieved in HSC, we transplanted one million BM mononuclear cells obtained from primary mice at weeks 4, 6 and 8 post-transplantation to secondary recipients. GFP+ human CD45+ cells were detected in secondary hosts 6 weeks after transplantation (Wk 4, 14.93±14.63, n=3; Wk 6, 0.87±0.44,n=3; Wk 8, 1.1±0.83,n=3). Engraftment of Bcr-Abl expressing cells in secondary hosts was confirmed by RT-PCR. In conclusion, we show that retroviral transduction of human CD34+ cells results in expression of the p210Bcr-Abl gene in cells capable of long-term multi-lineage engraftment of NSG mice and engraftment of secondary hosts, indicating the utility of this approach to study Bcr-Abl-mediated transformation of human HSC. Mice receiving transduced HSC develop progressive splenomegaly and increasing human cell engraftment in BM, but do not show selective proliferation of myeloid cells up to 20 weeks after transplantation. These results are in contrast to the vigorous myeloid progenitor proliferation seen when Bcr-Abl transduced human CD34+ cells are cultured in vitro, and the efficient and rapid induction of a myeloproliferative disease seen in mice transplanted with p210Bcr-Abl-transduced murine BM cells. Development of a myeloproliferative disorder following Bcr-Abl-mediated stem cell transformation may therefore be dependent on specific microenvironmental factors not available in the mouse host or may require the acquisition of additional abnormalities in Bcr-Abl expressing HSC.

An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259

Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of Hck and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of Hck by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (Hck-T338M). This substitution reduced Hck sensitivity to A-419259 by more than 30-fold without significantly affecting kinase activity in vitro. Expression of Hck-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of Hck-T338M kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type Hck retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34(+) CML cells with equal potency to imatinib. These data suggest that Hck has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.

Leukemogenic properties of NUP98-PMX1 are linked to NUP98 and homeodomain sequence functions but not to binding properties of PMX1 to serum response factor

PMX1 is a member of a non-clustered homeobox gene family, not normally expressed in hematopoietic cells, and first identified for its role in enhancing the binding of the serum response factor (SRF) to the serum responsive element (SRE). PMX1 has never been linked to leukemia on its own, raising the possibility of unique mechanisms underlying the oncogenicity of NUP98-PMX1. To elucidate the leukemogenic potential of NUP98-PMX1, we compared the effects of PMX1 and NUP98-PMX1 and, through strategic mutations, the involvement of the SRE in NUP98-PMX1-mediated leukemia. NUP98-PMX1, but not PMX1, had potent ability to impair differentiation, promote proliferation of myeloid progenitors, induce lethal myeloproliferative disease and to activate a number of genes previously linked to leukemic stem cells. Similar to NUP98-HOX fusions, the transforming potential of NUP98-PMX1 required the NUP98 portion and DNA-binding capability of the PMX1 homeodomain and collaborated with Meis1 to induce more rapid onset myeloproliferative-like myeloid leukemia. The transforming activity of NUP98-PMX1 was independent of its ability to interact with SRF. These findings provide novel evidence of the contributory role of the NUP98 sequence in conferring leukemogenic properties on a partner gene and point to common leukemogenic pathways for NUP98-PMX1 and NUP98-clustered HOX fusions.

K313dup is a recurrent CEBPA mutation in de novo acute myeloid leukemia (AML)

The CEBPA gene codes for a transcription factor that has a pivotal role in controlling proliferation and differentiation of myeloid progenitors. Acquired CEBPA mutations have been found in acute myeloid leukemias (AML) with a good prognosis, and most of these patients have a normal karyotype. In this paper, we report four cases that displayed the same K313dup in the CEBPA gene. All four had an AML-M1 with CD7 positivity and T-cell receptor gamma chain (TCR-gamma) rearrangement. This mutation could represent nearly 10% of all CEBPA mutations described to date. K313dup disappeared in samples from patients in complete remission. In transfected cells, the K313dup mutant had reduced protein stability with respect to the wild-type protein. K313dup seems to be selected in leukemic cells, and its frequency in other AML series could be determined using the screening method reported in this paper.

Eena Promotes Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation in Zebrafish

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena- deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, whereas eena lacking the Src homology 3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of the myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.

Requirement of c-Myb for p210BCR/ABL-dependent transformation of hematopoietic progenitors and leukemogenesis

AbstractThe c-Myb gene encodes a transcription factor required for proliferation and survival of normal myeloid progenitors and leukemic blast cells. Targeting of c-Myb by antisense oligodeoxynucleotides has suggested that myeloid leukemia blasts (including chronic myelogenous leukemia [CML]–blast crisis cells) rely on c-Myb expression more than normal progenitors, but a genetic approach to assess the requirement of c-Myb by p210BCR/ABL-transformed hematopoietic progenitors has not been taken. We show here that loss of a c-Myb allele had modest effects (20%-28% decrease) on colony formation of nontransduced progenitors, while the effect on p210BCR/ABL-expressing Lin− Sca-1+ and Lin− Sca-1+Kit+ cells was more pronounced (50%-80% decrease). Using a model of CML-blast crisis, mice (n = 14) injected with p210BCR/ABL-transduced p53−/−c-Mybw/w marrow cells developed leukemia rapidly and had a median survival of 26 days, while only 67% of mice (n = 12) injected with p210BCR/ABL-transduced p53−/−c-Mybw/d marrow cells died of leukemia with a median survival of 96 days. p210BCR/ABL-transduced c-Mybw/w and c-Mybw/d marrow progenitors expressed similar levels of the c-Myb–regulated genes c-Myc and cyclin B1, while those of Bcl-2 were reduced. However, ectopic Bcl-2 expression did not enhance colony formation of p210BCR/ABL-transduced c-Mybw/d Lin−Sca-1+Kit+ cells. Together, these studies support the requirement of c-Myb for p210BCR/ABL-dependent leukemogenesis.

Modeling of C/EBPα Mutant Acute Myeloid Leukemia Reveals a Common Expression Signature of Committed Myeloid Leukemia-Initiating Cells

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

The receptor for Granulocyte-colony stimulating factor (G-CSF) is expressed in radial glia during development of the nervous system

BackgroundGranulocyte colony-stimulating (G-CSF) factor is a well-known hematopoietic growth factor stimulating the proliferation and differentiation of myeloid progenitors. Recently, we uncovered that G-CSF acts also as a neuronal growth factor in the brain, which promotes adult neural precursor differentiation and enhances regeneration of the brain after insults. In adults, the receptor for G-CSF is predominantly expressed in neurons in many brain areas. We also described expression in neurogenic regions of the adult brain, such as the subventricular zone and the subgranular layer of the dentate gyrus. In addition, we found close co-localization of the G-CSF receptor and its ligand G-CSF. Here we have conducted a systematic expression analysis of G-CSF receptor and its ligand in the developing embryo.ResultsOutside the central nervous system (CNS) we found G-CSF receptor expression in blood vessels, muscles and their respective precursors and neurons. The expression of the G-CSF receptor in the developing CNS was most prominent in radial glia cells.ConclusionOur data imply that in addition to the function of G-CSF and its receptor in adult neurogenesis, this system also has a role in embryonic neurogenesis and nervous system development.

Regulation of progenitor cell proliferation and granulocyte function by microRNA-223

MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programmes. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 (also called Mirn223) mutant mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors. We show that Mef2c, a transcription factor that promotes myeloid progenitor proliferation, is a target of miR-223, and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. As a consequence of this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction after endotoxin challenge. Our data support a model in which miR-223 acts as a fine-tuner of granulocyte production and the inflammatory response.

Deletion of HoxA Genes Inhibits Proliferation and Differentiation of Myeloid Progenitors.

Mutants for single homeobox genes are showing neglible to mild phenotypes in the hematopoietic compartment. This is likely due to functional redundancy between Hox genes that has been demonstrated in embryonic development by compound mutant models. Interestingly, our previous data showed that hematopoietic stem cells (HSCs) lacking the majority of the HoxB genes (Hoxb1-Hoxb9) remained fully competent in replenishment of the hematopoietic compartment (Bijl, 2006). In addition we reported the expression of the majority of the HoxA cluster genes in HSC enriched E14.5 fetal liver fractions at levels at least one log higher than for HoxB cluster genes, suggesting that not HoxB but HoxA genes have a physiological role in hematopoiesis at that stage in the ontogeny. To investigate further whether HoxA genes are required for definitive hematopoiesis we used a conditional knock-out mouse model, in which the HoxA cluster was flanked by loxP sites (HoxAflox/flox). To excise the HoxA genes Cre recombinase in conjunction with GFP was retrovirally expressed in HoxAflox/flox fetal liver cells (Ly5.2). Flow cytometry showed transduction efficiencies in total fetal liver of 40% for Cre-GFP and 60% for MSCV-GFP control vector. Transduced cell composites were both plated in myeloid colony forming unit assays and transplanted in congenic (Ly5.1) mice to evaluate the transduction rate in progenitor populations and their capacity to proliferate and differentiate. Cre+ progenitor cells were able to grow out in colonies, and cultures showed a transduction efficiency of 69% for Cre-GFP and 99% for control vectors at the level of progenitors. The excision of the HoxA locus was confirmed by PCR for two Cre-GFP colonies. However, Cre-GFP+ colonies were much smaller than control colonies, indicating a defect in the proliferation potential of Cre+ myeloid progenitors. Further expansion of GM colonies in liquid culture was less efficient for Cre+ (1 out of 15), than for controls (5 out of 24), underlining a major defect in proliferation of early myeloid progenitors with granulocytic and monocytic differentiation potential in the absence of HoxA genes. All the colony types (GEMM, GM-CFU, G-CFU, M-CFU and BFU-E) were present in the Cre+ culture, but GM-CFUs and M-CFUs were proportional less represented than in control cultures (17% vs. 26% and 40% vs. 48%, respectively), indicating that HoxA genes have a function in early myeloid differentiation. Moreover, all colonies were in general poorly differentiated, showing that myeloid differentiation is sensible to reduced HoxA levels particularly in progenitors for the granulocytic/monocytic lineage. The capacity of HoxA−/− progenitors to induce small colonies might be due to some residual levels at the time of excision or in the case of mature progenitors a reduced requirement for HoxA genes, which is in agreement with the decreased expression of Hox genes with maturation of blood cells. No progeny of Cre-GFP+ cells was observed in irradiated recipients 4 weeks post-transplantation, which demonstrate complete failure of HoxA−/− progenitors to repopulate. However, it is too early to conclude whether deletion of HoxA genes affects the activity of HSCs. Altogether our in vitro and in vivo data show that HoxA genes are essential for the proliferation and differentiation of early myeloid progenitors that seems not to be compensated by other Hox gene members. The effect of HoxA deletion on HSC activity is currently under investigation.

Expression of G-CSF Receptor during Embryonic Development in a Csf3r-Cre Knock-In Mouse Model.

G-CSF is the major regulator of neutrophil development and controls the proliferation, differentiation and survival of myeloid progenitors in both steady state and “emergency” granulopoiesis. More recently however, novel activities of the G-CSF/G-CSF receptor (CSF3R) axis have been suggested, especially after severe forms of tissue damage. For instance, upon myocardial infarction, G-CSF was shown to protect cardiomyocytes from apoptosis, reducing the extent of myocardial damage. Likewise, in ischemic stroke models, G-CSF treatment reduced infarct volumes by promoting neuronal survival and differentiation of neural stem cells in the brain. Finally, G-CSF has been suggested to promote hepatic regeneration after severe liver damage by directly stimulating the proliferation of oval cells. To study whether and to what extent CSF3R expression in damaged tissues reflects reactivation of a normal development program, we generated a csf3r-Cre knock-in model. To this end, the csf3r locus was targeted with a construct containing the cre gene and a puromycin selection cassette flanked by a 2.7 kb fragment of the csf3r 5′UTR and a 4 kb fragment directly downstream of the initiation codon of csf3r. Correct insertion of the cre gene was confirmed by Southern blotting and sequence analysis and subsequent crossing with FLP deleter mice resulted in successful germ line transmission and deletion of the puromycin cassette. These mice were crossed with the R26R LacZ reporter mouse and β-galactosidase (β-gal) activity was examined by FACS and histochemical analysis. At E9.5, no β-gal activity was detected, indicating that csf3r is not expressed at that stage of development. In contrast, in E12.5 embryos, β-gal staining was observed in fetal liver, heart, kidney, brain and intestine. Analysis of adult tissues showed that cardiomyocytes were β-gal positive in a mosaic pattern. A similar mosaicism was observed in liver, kidney and the intestines. In liver both hepatocytes and vascular endothelial cells stained positive, whereas in the kidney β-gal staining was detected in vascular endothelium, glomeruli and afferent and efferent vessels. In ileum and colon, β-gal activity was mainly seen in the stem and progenitor cell-containing crypts and the developing progeny migrating upwards along the villus. In bone marrow, high levels (>40%) of β-gal activity were detected in Gr1+ and CD11b+ myeloid cells, CD19+ (B) and CD3ε+ (T) lymphoid cells, NK cells and CD11c+ dendritic cells, indicating that G-CSFR was expressed in a common progenitor of these cell types. In agreement with this, β-gal activity was also detected in the primitive LSK population. In conclusion, our data indicate that CSF3R is expressed in both hematopoietic and multiple non-hematopoietic organs from embryonic development stage E12.5 onward. This may have important ramifications for a more wide-spread application of G-CSF in tissue regeneration.

Serial Analysis of Hematopoietic Progenitors in Mpl−/− Mice.

Thrombopoietin (TPO) is the pivotal regulator of megakaryocytopoiesis but also an important factor for early multipotent hematopoietic progenitors. Loss-of-function mutations in the human MPL gene coding for the TPO receptor are the cause of congenital amegakaryocytic thrombocytopenia (CAMT), a rare inherited bone marrow failure syndrome presenting as isolated thrombocytopenia at birth and progressing to aplastic pancytopenia during the first years of life. In contrast, Mpl−/− mice although exhibiting thrombocytopenia and severely reduced numbers of early hematopoietic progenitors do not develop any signs of pancytopenia or generalized bone marrow failure during lifetime. To identify parallels and differences in the impact of TPO on hematopoietic development between mice and men we serially analyzed subpopulations of hematopoietic cells from early progenitors to mature peripheral blood cells from Mpl−/− mice (kindly provided by Warren Alexander, WEHI, Melbourne, Australia) over a period of 24 months. We determined frequency, clonogenicity and proliferative potential of hematopoietic progenitors from different hematopoietic tissues. Compared to wild type mice we found a strong reduction of multipotent stem cells (HSC), of common myeloid progenitors (CMP), of megakaryocyte/erythrocyte lineage restricted progenitors (MEP) but also of common lymphoid progenitors (CLP) in Mpl−/− mice. We did not find any significant changes in the frequency of these cells over a period of 24 months. For the CMP and MEP subpopulations of myeloid progenitors we found not only a decreased frequency but also a reduced clonogenicity and proliferative capacity. Interestingly, these functional restrictions significantly increased significantly during aging, a finding that was similar to our results obtained with myeloid progenitors from CAMT patients. Impaired proliferation of myeloid progenitors rather than a reduced number could be the reason for development of pancytopenia in CAMT patients.

A phase II trial of tipifarnib in myelofibrosis: primary, post-polycythemia vera and post-essential thrombocythemia

Patients with primary myelofibrosis (PMF) or post-polycythemia vera or post-essential thrombocythemia myelofibrosis (post-PV/ET MF) have limited therapeutic options. The farnesyltransferase-inhibitor tipifarnib inhibits in vitro proliferation of myeloid progenitors from such patients. In the current phase II clinical trial, single-agent oral tipifarnib (300 mg twice daily x 21 of 28 days) was given to 34 symptomatic patients with either PMF (n=28) or post-PV/ET MF (n=6). Median time to discontinuation of protocol therapy was 4.6 months; reasons for early termination (n=19; 56%) included disease progression (21%) and adverse drug effects (18%). Toxicities (>/=grade 3) included myelosuppression (n=16), neuropathy (n=2), fatigue (n=1), rash (n=1) and hyponatremia (n=1). Response rate was 33% for hepatosplenomegaly and 38% for transfusion-requiring anemia. No favorable changes occurred in bone marrow fibrosis, angiogenesis or cytogenetic status. Pre- and post-treatment patient sample analysis for in vitro myeloid colony growth revealed substantial reduction in the latter. Clinical response did not correlate with either degree of colony growth, measurable decrease in quantitative JAK2(V617F) levels or tipifarnib IC(50) values (median 11.8 nM) seen in pretreatment samples. The current study indicates both in vitro and in vivo tipifarnib activity in PMF and post-PV/ET MF.

Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1-C/EBPα interaction

Interactions between the cell cycle machinery and transcription factors play a central role in coordinating terminal differentiation and proliferation arrest. We here show that cyclin-dependent kinase 6 (Cdk6) is specifically expressed in proliferating hematopoietic progenitor cells, and that Cdk6 inhibits transcriptional activation by Runx1, but not C/EBPalpha or PU.1. Cdk6 inhibits Runx1 activity by binding to the runt domain of Runx1, interfering with Runx1 DNA binding and Runx1-C/EBPalpha interaction. Cdk6 expression increased myeloid progenitor proliferation, and inhibited myeloid lineage-specific gene expression and terminal differentiation in vitro and in vivo. These effects of Cdk6 did not require Cdk6 kinase activity. Cdk6-mediated inhibition of granulocytic differentiation could be reversed by excess Runx1, consistent with Runx1 being the major target for Cdk6. We propose that Cdk6 downregulation in myeloid progenitors releases Runx1 from Cdk6 inhibition, thereby allowing terminal differentiation. Since Runx transcription factors play central roles in hematopoietic, neuronal and osteogenic lineages, this novel, noncanonical Cdk6 function may control terminal differentiation in multiple tissues and cell types.