Myeloid Progenitor Compartment Research Articles

The Cell Polarity PTK7 Receptor Is Expressed in Myeloid Cells and Acts as a Modulator of the Chemotherapeutic Response in AML Patients.

Abstract 1571Poster Board I-596The pseudo tyrosine kinase receptor 7 (PTK7) is an orphean tyrosine kinase receptor assigned to the planar cell polarity pathway (PCP). It has been recently described and plays a major role during embryogenesis and epithelial tissue organisation. To date there is no report in the litterature considering a potential implication in hematopoiesis. In silico and in vitro analysis found that PTK7 was also expressed in normal myeloid progenitors and CD34+ CD38- bone marrow cells in humans. Preliminary results from our team showed that PTK7 was also expressed in various leukemic cell lines such Jurkat, TF-1 or KG-1a. We decided to perform a wide range multicolour immunophenotyping screen on patients with acute myeloid leukemia (AML) at diagnosis and to investigate the role of PTK7 in AML in vitro. More than 250 patient samples were evaluated and we demonstrated that PTK7 was largely expressed in AML as 72% of the samples were PTK7 positive. Its expression mostly correlates with granulocytic lineage differentiation. PTK7 expression was associated with a lower WBC count at diagnosis and a lower frequency of extramedullary disease whatever was FAB subtype. Interestingly, PTK7 expression was associated with some cytogenetic subgroups including CBF-AML and APL. There was no correlation with molecular subgroups (i.e. FLT3-ITD/NPM1/CEBPA status). Overall Survival and Relapse Free Survival were evaluated in non-APL patients treated with induction chemo (n=182). Patients with PTK7 positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced Relapse Free Survival in a multivariate analysis model integrating all pre treatment variables (2 year probability of RFS= 29% vs 66% for PTK7 negative patients, p= 0.003). Forrest plot analysis showed that the negative impact of PTK7 expression was the most significant in intermediate cytogenetic risk subgroup and when PTK7 was aberrantly expressed in M4-M5 FAB subtypes. There was no demonstrated impact on CR. In cultured cells, expression of PTK7 promotes leukemia cell migration, cell survival and resistance to anthracyclin-induced apoptosis. There was no effect of PTK7 expression on cell proliferation in tritiated thymidine assay. In the absence of known inhibitor of PTK7, we produced a soluble recombinant PTK7-Fc protein that efficiently competes for PTK7 functions in cell migration and survival assays in cell lines and primary AML samples. These data were confirmed using a shRNA strategy. We conclude that PTK7 is a PCP component expressed in the myeloid progenitor compartment that conveys promigratory and anti-apoptotic signal to leukemia cells. Its use as a potential biomarker or therapeutical target should be investigated. DisclosuresNo relevant conflicts of interest to declare.

Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses.

Abstract 2510Poster Board II-487Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. We have observed that introduction of activated STAT5A in human stem/progenitor cells enhances long-term stem cell self-renewal, while lentiviral downmodulation of STAT5 expression in both normal as well as primary leukemic CD34+ cells impairs long-term growth and self-renewal. Many cytokines that act on the immature human stem cell compartment are known to be able to activate STAT5, such as TPO and SCF. Yet, little is known about the specificity and kinetics of STAT5 signaling in response to early-acting and lineage-restricted cytokines in specifically defined stem cell and progenitor compartments, particularly in the case of acute myeloid leukemia. We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at the single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34+CD38− hematopoietic stem cells (HSCs). EPO and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF, as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34+ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. A number of different types of responses were observed, being a) a strong STAT5 activation induced by TPO only; b) a strong STAT5 activation induced by IL3 and GM-CSF, but not TPO; c) STAT5 activation induced by various cytokines; and d) constitutive STAT5 phosphorylation that could not significantly be further induced by cytokines. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34+ cells on stroma. In most examined cases multiple cytokines acted in an additive fashion in inducing long-term growth of primary AML CD34+ cells in bone marrow stromal cocultures, and in only 1 out of 10 cases cytokine-independent growth was observed. In none of the cases only IL3 or TPO were sufficient to induced long-term expansion of primary AML CD34+ cells. In conclusion, our study demonstrates variable cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

FLT3 Internal Tandem Duplication (ITD) Mutations Disrupt Homeostasis in Hematopoietic Stem Cells.

Abstract 1420Poster Board I-443As one of the most common genetic alterations found in acute myeloid leukemia (AML), constitutive activation of the FMS-like tyrosine kinase (FLT3) has provided a promising candidate for small molecule targeted therapy. However, the results of FLT3 inhibitor monotherapy trials indicate FLT3 inhibition alone is insufficient to induce consistent and durable responses. Moreover, after an initial response, many patients relapse, suggesting that leukemia-initiating stem cells may be escaping inhibitor-induced cytotoxicity. Currently, the exact stage at which activating mutations in FLT3 occur during transformation is unknown. While FLT3 knockout mice have minor defects in hematopoiesis, very little is known about either the effect of FLT3 activating mutations on normal hematopoietic stem cells or the contribution of FLT3 activation to leukemogenesis. Thus, in order to better understand the underlying molecular mechanisms of transformation and to identify novel targets for treatment of AML, the role of FLT3 activating mutations in hematopoietic stem cells (HSCs) is of great interest.To study the natural stem cell reservoir and other populations that may escape inhibition, our laboratory has developed a knock-in mouse model in which the FLT3/ITD mutation (an internal tandem duplication correlated with poor prognosis in patients) has been introduced under the endogenous promoter, resulting in myeloproliferative disease (MPD). Conventional transplantation using unfractionated or lineage-depleted marrow from FLT3/ITD mice failed to fully engraft or recapitulate disease, suggesting a HSC defect. Thus, in order to identify a compartment enriched for MPD-initiating cells, several cell surface marker-defined hematopoietic populations were transplanted and compared for engraftment and disease recapitulation. Lineage negative (LIN-), KSL (KIT+SCA+LIN-), MPP (KSL CD34+FLT3+), and ST-HSC (KSL CD34+FLT3-) cells sorted from FLT3/ITD bone marrow all had significantly reduced reconstitution capacity compared to the same compartment from WT littermates. In contrast, highly purified LT-HSCs (KSL CD34-FLT3-) generated equivalent engraftment whether from WT or ITD bone marrow. Furthermore, we measured Hoechst dye efflux in WT and ITD bone marrow to examine side population (SP) cells, known to be enriched in HSC activity, and found that FLT3/ITD mice displayed five-fold fewer SP cells. In addition, bone marrow from FLT3/ITD mice showed a ten-fold decrease in SLAM-defined stem cell frequency (LIN-CD48-CD41-CD150+). 500 sorted SLAM cells from either WT or FLT3/ITD mice were sufficient to fully reconstitute a transplant recipient, demonstrating an equivalent engraftment capacity within this HSC-enriched compartment. Moreover, the MPD phenotype was successfully recapitulated in primary transplants of FLT3/ITD SLAM cells as characterized by an increase in myeloid progenitors, expansion of the LIN- fraction, enlarged spleens and depletion of the SLAM compartment compared to WT SLAM transplant recipients.Classically defined as a class II oncogene, FLT3 activating mutations have been shown to drive proliferation in cells harboring the mutation. To investigate whether FLT3/ITD drives proliferation in the most primitive hematopoietic compartments, BrdU incorporation was examined in FLT3/ITD hematopoietic stem and progenitor-enriched subsets. While myeloid progenitor compartments showed a decrease in proliferation as compared to WT littermates, the FLT3/ITD SLAM and KSL compartments had an increased percentage of BrdU-incorporating cells. Altogether, our data suggests a role for FLT3/ITD in driving normally quiescent HSCs to proliferate, thereby depleting the pool of primitive HSCs. In this model, HSC depletion coupled to rapid expansion in progenitor cell numbers leads to perturbation of normal hematopoiesis giving rise to a myeloproliferative disease. Disclosures:No relevant conflicts of interest to declare.

IL-1R Type I-Dependent Hemopoietic Stem Cell Proliferation Is Necessary for Inflammatory Granulopoiesis and Reactive Neutrophilia

Infections and inflammation trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as emergency granulopoiesis. The intrinsic factors that drive reactive neutrophilias and emergency granulopoiesis have been inferred but not demonstrated. Here, we show that alum cannot elicit reactive neutrophilias in IL-1R type I (IL-1RI)(-/-) mice, whereas other inflammatory responses, including eosinophilia and Ab production, remain intact. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors and, surprisingly, multipotent progenitors and hematopoietic stem cells (HSC). Indeed, HSC and multipotent progenitor proliferative responses were most suppressed in IL-1RI(-/-) mice, suggesting a critical role for their proliferation in inflammatory granulopoiesis. Whereas IL-1 drives increased HSC proliferation directly in vitro, IL-1RI expression by radiation-resistant host cells was both necessary and sufficient for alum-induced HSC, multipotent progenitor, and granulocyte/macrophage progenitor proliferation and reactive neutrophilias in radiation chimeric mice. Thus, IL-1 plays a necessary, but indirect, role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.

Abnormalities in the myeloid progenitor compartment in Down syndrome fetal liver precede acquisition of GATA1 mutations

AbstractDown syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal–truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyte-erythroid progenitor frequency was increased (55.9% ± 4% vs 17.1% ± 3%, CD34+CD38+ cells; P < .001) with common myeloid progenitors (19.6% ± 2% vs 44.0% ± 7%) and granulocyte-monocyte (GM) progenitors (15.8% ± 4% vs 34.5% ± 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34+ cells was markedly increased (78% ± 7% vs 15% ± 3%) affecting megakaryocyte-erythroid (∼ 7-fold higher) and GM and colony-forming unit–granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to GATA1 mutations in DS-AMKL and DS-associated transient myeloproliferative disorder.

5′ Azacitidine in Combination with Valproic Acid Induces Complete Remissions in Patients with Advanced Acute Myeloid Leukaemia but Does Not Eradicate Clonal Leukaemic Stem/Progenitor Cells.

Demethylating drugs (e.g. 5′ Azacitidine) and histone deacetylation inhibitors are a relatively new class of clinically active agents for acute myeloid leukaemia (AML) and high-risk myelodysplasia (MDS) that have engendered a lot of interest. However, their mechanisms of action (in terms of achieving a clinical response), and how best to use them, both remain unclear. These agents rarely cure patients; in most cases the disease relapses. To better understand the impact of these agents on haemopoiesis, we have treated 45 patients with high risk AML/MDS (30 relapsed/refractory AML, 6 de novo AML, 9 high risk MDS) with azacitidine (75 mg/m2 5 days every 28 days), sodium valproate, all-trans retinoic acid and theophylline and correlated clinical response with immunophenotypic and clonogenic analysis of committed and multi-potent leukemic stem/progenitor cells. The median age of the cohort was 66 years (range 32–85 years). Clinical responses were assessed using Cheson criteria. Responses were observed in 15 patients; 7 achieved a complete remission (CR) or CRi and 8 a partial response (>50% reduction in bone marrow blasts). The median time to achievement of maximal response was 2 cycles (range 1–6). Cytogenetic data in 40/45 patients studied showed abnormalities in 60% (24/40) of patients of which 50% (12/24) were adverse risk. 40% (6/15) patients achieving CR, CRi or PR had adverse risk cytogenetics. The most common grade 3/4 haematological toxicities were thrombocytopenia (51%), leukopenia (42%) and neutropenia (42%). Common non-haematologic toxicities were fatigue (31%), neutropenic sepsis (20%), chest infection (16%) and diarrhoea (13%). Detailed immunophenotypic analysis, clonal assays (methyl cellulose and cobblestone-area forming colony assays, CAFC) and FISH analysis were performed on stem cell enriched (CD34+CD38−), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte erythroid progenitor (MEP) compartments at diagnosis and post-treatment. In all diagnostic samples there were abnormal immunophenotypic CD34+CD38-, CMP, GMP and MEP (myeloid progenitor) compartments. There was an anticipated failure of normal colony and CAFC growth from sorted CD34+CD38- and myeloid progenitor populations. In responding, but not non-responding, patients there was partial or complete restoration of normal immunophenotypic populations accompanied by normal haemopoietic colony output. However, we extended these analyses to look at the kinetics of change in clonally abnormal cells that remain after treatment in total bone marrow mononuclear cells and FACS sorted cells CD34+CD38- and myeloid progenitor populations from one informative AML patient with an abnormal karyotype at diagnosis, who had a complete morphological response. At diagnosis, cytogenetic FISH analysis on sorted cells showed that 90–100% cells were clonally abnormal in the CD34+CD38- and myeloid progenitor compartments. At the beginning of cycle 3, there wascomplete haematological response (normal blood count and bone marrow myelogram and morphology):karyotypic remission in bone marrow mononuclear cells (50 cells examined);restoration of normal colony output; andnormal immunophenotypic pattern of stem/progenitor populations.However, in sorted stem/progenitor populations, clonally abnormal cells were still detected by FISH at ~15% level. These abnormal cells provide a cellular substrate for future loss of complete clinical response. This was seen by cycle 6. Now the patient developedperipheral cytopenias;reduction in normal colony output;increased bone marrow dysplasia but no excess of blasts.The bone marrow mononuclear karyotype was still normal. However, now there was an increase to 100% clonally abnormal cells in the GMP compartment whereas the CD34+CD38- compartment there were still 15% clonally abnormal cells. Though information from additional patients under study will be critical to understand the generality of these findings, these data provide the first insights into the impact of treatment and the cellular kinetics of relapse. In conclusion, this is the first study to document the clinical and biological impact on stem/progenitor and peripheral haemopoiesis of this class of agent, or indeed of any drug in AML and MDS. The findings from studies like this will have general implications on how to assess the clinical and biological impact of therapies in AML and MDS.

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

Expression of PML-RARα by the Murine PML Locus Leads to Myeloid Self-Renewal, Clonal Expansion and Morphologic Promyelocytic Leukemia.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation that leads to expression of a fusion protein, PML-RARα, and haploinsufficiency for both RARα and PML. We have generated a novel murine model of APL using homologous recombination to place a pathogenic human PML-RARα cDNA into the murine PML locus (mPML-PRflox). Expression of PML-RARα is initially prevented by stop codons within the loxP-flanked PGK-neo cassette. This new model recapitulates key elements of human APL lacking in other models; it provides PML locus appropriate regulation of PML-RARα expression, haploinsufficiency of PML, and somatic acquisition of the fusion protein.We have exposed mPML-PRflox mice to a conditionally-active Cre transgene (ER-T2- Cre, which activates Cre only during Tamoxifen treatment), and a compartmentally restricted Cre transgene (Lysozyme M-Cre, which has low activity in early myeloid precursors and increasing activity with myeloid differentiation). Bone marrow and spleen cells doubly heterozygous (DH) for mPML-PRflox and an activated Cre allele express PML-RARα mRNA, display neutrophil POD disruption typical of PML-RARα activity, and exhibit a shift in myelopoiesis toward CFU-G formation. We found that expression of PML-RARα following transient ER-T2-Cre activation leads to myeloid self-renewal ex vivo and clonal expansion in vivo. Bone marrow cells from DH mPML-PRflox/ER-T2- Cre mice exposed to Tamoxifen could be serially replated in methylcellulose. With successive replating, the proportion of cells carrying a floxed PML-RARα allele increased from 30% in bone marrow cells to 95% following the third replating; DH mPML-PRflox/ ER-T2-Cre bone marrow cells that were not exposed to Tamoxifen, and bone marrow cells from wild type mice, could not be serially replated. Cells bearing a floxed PML-RARα allele also expanded in vivo. In DH mPML-PRflox/ER-T2-Cre mice, a single dose of Tamoxifen (4 mg) resulted in 5% of peripheral blood cells carrying a floxed PML-RARα allele on day 7, but this population expanded progressively to 40% on day 80 without further Tamoxifen exposure (n=5). Five doses of Tamoxifen (4 mg) lead to 20% peripheral blood cells caring a floxed PML-RARα allele on day 18, and this increased progressively to 80% on day 98 (n=4). Peripheral blood of DH mPML-PRflox/ER-T2-Cre mice unexposed to Tamoxifen carried undetectable or trace numbers of cells with a floxed PML-RARα allele on day 80 (n=3). In contrast, myeloid progenitors from DH mPMLPRflox/ Lysozyme M-Cre mice did not display self-renewal ex vivo or an expansion of floxed cells with successive methylcellulose replating. DH mPML-PRflox/Lysozyme M-Cre mice did develop promyelocytic leukemia with long latency (14 months) and low penetrance (7%, n=3), which could be transplanted to secondary recipients. These tumors displayed a high percentage of floxed PML-RARα alleles in peripheral blood, spleen and bone marrow cells (range 50 – 95%) and possessed 4.5 fold higher expression levels of PML-RARα mRNA than our previously characterized Cathepsin G PML-RARα knock-in tumors. Importantly, non-leukemic,18 month-old DH mPML-PRflox/Lysozyme M-Cre mice displayed little evidence of PML-RARα dependent clonal expansion. In these mice, peripheral blood cells and spleen cells maintained low levels of the floxed PML-RARα allele (10–20%), equivalent to 6 week-old mice (n=36).These data suggest that PML-RARα expression by the murine PML locus leads directly to a myeloid self-renewal program and clonal expansion. Since Lysozyme M-Cre is expressed at low levels in early myeloid progenitors, the low penetrance of leukemia and rare clonal expansion in DH mPML-PRflox/Lysozyme M-Cre animals suggests that APL leukemogenesis may require PML-RARα expression in an early myeloid progenitor compartment, rather than a late compartment.

Molecular Signature of Distinct CD34+ Hematopoietic Stem and Progenitor Cell Subsets of Patients with CML in Chronic Phase.

During the last decade, chronic myeloid leukemia (CML) has been mainly characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the protooncogene BCR-ABL. This constitutively active tyrosine kinase is widely considered as the cause of the disease. Even though BCR-ABL transcripts are found in every dividing hematopoietic cell and thus, the disease is likely to originate from a primitive stem cell, the “cell of origin” is still a matter of debate. Despite the active “leukemia stem cell” discussion, very few characteristics of the “cancer stem cell” are established to date. In order to get further molecular insights into CML stem and progenitor cells, we examined CD34+ cell subsets obtained from bone marrow of 7 patients with CML in chronic phase in comparison with 5 healthy volunteers. CD34+ cells were immunomagnetically selected and high-speed cell sorting of lineage-negative, CD34+, CD38−, hematopoietic stem cells and myeloid progenitors was performed. Progenitors were further subdivided by anti-IL-3Ralpha and anti-CD45RA staining. Following RNA extraction, a two-cycle amplification procedure was used to generate cDNA for the hybridization with Affymetrix U133A2.0 arrays. After performing smoothening spline normalization, we applied the perfect match-mismatch difference model algorithm to calculate expression values (dChip). Hierarchical cluster analysis was performed using a correlation based centroid linkage algorithm. Hereby we could discriminate the HSCs, CMPs, and MEP subsets. Corroboration of RNA expression was performed by real-time RT-PCR for selected genes. Comparing the HSC subsets of CML patients with healthy controls we found 98 differentially expressed genes. 87 genes had a lower expression level in CML HSCs whereas 11 genes had a higher one. Among the downregulated genes in CML were transcriptions factors involved in myelogenesis and proliferation and several adhesion molecules associated with homing and migration of the HSCs. On the other hand, the Leptin receptor and BCR-ABL downstream targets were found to be upregulated. Within the common myeloid progenitor (CMP) compartment 37 genes were significantly differentially regulated. Twenty genes had a higher expression level in CML CMPs, 17 genes were downlegulated. Hematopoietic cell-specific cell cycle inhibitor MS4A3 was among the significantly downregulated genes whereas genes of the retinoblastoma and E2F families as well as inhibitors of the Wnt-signaling pathway were upregulated. Looking at megakaryocte-erythrocyte progenitors (MEP) in CML, key mediators of G2-M cell cycle transition were downregulated indicating a lower proliferative capacity of this subset. No transcriptional differences have been observed between granulocyte-macrophage progenitors from CML patients and healthy volunteers. Interestingly, among all other subsets myeloperoxidase (MPO) was downregulated in the CML samples and the Leptin receptor was upregulated. Our results provide novel insights into the biology of CML and potentially provide the basis for the characterization of a candidate CML stem cell.

FLT3 Mutations Confer Enhanced Proliferation and Survival Properties to Multipotent Progenitors in a Murine Model of Chronic Myelomonocytic Leukemia

Despite their known transforming properties, the effects of leukemogenic FLT3-ITD mutations on hematopoietic stem and multipotent progenitor cells and on hematopoietic differentiation are not well understood. We report a mouse model harboring an ITD in the murine Flt3 locus that develops myeloproliferative disease resembling CMML and further identified FLT3-ITD mutations in a subset of human CMML. These findings correlated with an increase in number, cell cycling, and survival of multipotent stem and progenitor cells in an ITD dose-dependent manner in animals that exhibited alterations within their myeloid progenitor compartments and a block in normal B cell development. This model provides insights into the consequences of constitutive signaling by an oncogenic tyrosine kinase on hematopoietic progenitor quiescence, function, and cell fate.

Elucidation of the Phenotypic, Functional, and Molecular Topography of a Myeloerythroid Progenitor Cell Hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

The role of interleukin 6 in megakaryocyte formation, megakaryocyte development and platelet production.

Megakaryocytopoiesis is the cellular amplification and differentiation of precursors into immature megakaryocytes, and the cytoplasmic maturation of these megakaryocytes, a process terminating in the release of platelets into the circulation. Interleukin 6 (IL-6) stimulates megakaryocytopoiesis in the bone marrow, increasing platelet numbers in the circulation. IL-6 alone is poorly active on the growth of stem cell populations, but acts in synergy with stem cell factor (c-kit ligand) to expand the committed myeloid progenitor compartments but not the megakaryocyte progenitors. IL-6 has a direct action on megakaryocyte progenitors but only in synergy with low doses of interleukin 3 (IL-3), increasing the number of immature megakaryocytes and enhancing the processes of development into mature megakaryocytes. IL-6 is about 10 times more active on megakaryocytes than on megakaryocyte progenitors in cell culture. It is active alone and will stimulate increases in cell size and DNA content. IL-6 does not appear to stimulate the process of platelet release. IL-6 is found in bone marrow, in both macrophage subsets and megakaryocytes, indicating that it may be an important physiological regulator of both paracrinal (microenvironmental) and autocrinal mechanisms controlling megakaryocyte development in bone marrow.

The Retinoblastoma Binding Protein RBP2 Is an H3K4 Demethylase

Changes in histone methylation status regulate chromatin structure and DNA-dependent processes such as transcription. Recent studies indicate that, analogous to other histone modifications, histone methylation is reversible. Retinoblastoma binding protein 2 (RBP2), a nuclear protein implicated in the regulation of transcription and differentiation by the retinoblastoma tumor suppressor protein, contains a JmjC domain recently defined as a histone demethylase signature motif. Here we report that RBP2 is a demethylase that specifically catalyzes demethylation on H3K4, whose methylation is normally associated with transcriptionally active genes. RBP2-/- mouse cells displayed enhanced transcription of certain cytokine genes, which, in the case of SDF1, was associated with increased H3K4 trimethylation. Furthermore, RBP2 specifically demethylated H3K4 in biochemical and cell-based assays. These studies provide mechanistic insights into transcriptional regulation by RBP2 and provide the first example of a mammalian enzyme capable of erasing trimethylated H3K4.

Rôle des seconds messagers lipidiques impliqués dans la réponse des cellules leucémiques aux médicaments anticancéreux

Simple clinical observation suggests that while anti-leukemia agents are efficient at eradicating blasts cells in terminal division, as illustrated, in the case of acute myeloid leukemia, by the high complete remission rate (70%); these agents are relatively inept at eliminating leukemic myeloid progenitors as suggested by the high level of recurrence. This interpretation underlines the apparently natural chemoresistance of cells which compose the myeloid leukemia progenitor compartment. Over the past few years, several studies have shown that similar cellular damage can lead to divers effects such as rapid apoptotic death, differed mitotic death, or a transitory cytostatic effect. Cell response to damage is regulated by a complex and highly regulated network of intracellular signals including cell death signals mediated by ceramide and cell survival signals mediated (at least in part) by diacylglycerol and phosphoinositide-3 phosphates. Cellular fate relies on the balance between these two signaling pathways. This hypothesis opens several prospects on pharmacological manipulation aimed at either favoring cell death or at conferring resistance to anti-cancer agents.

Megakaryocytopoiesis and platelet production: does stem cell factor play a role?

Megakaryocytopoiesis, resulting in the production and release of platelets, is a multistage procession of cellular differentiation and maturation which is regulated by a constellation of cytokines. Since thrombocytopenia is a frequent dose-limiting toxicity of chemotherapy, newly-identified cytokines have been actively investigated for their potential megakaryocyte/platelet-promoting properties. Stem cell factor (SCF, also known as mast cell growth factor, Steel factor or Kit ligand) has been found to synergize with GM-CSF, IL-6, IL-3, IL-11 or Epo to increase the numbers of megakaryocyte-containing colonies (i.e., CFU-Meg, BFU-Meg, CFU-GMM, CFU-GEMM). On the other hand, SCF increased the number of megakaryocytes per colony in the presence of IL-3, GM-CSF or IL-6. SCF also stimulated the proliferation of specific megakaryocytic cell lines (i.e., CMK, M-07e). SCF did not, however, alter megakaryocyte markers or increase cell ploidy. Thus, SCF appears to expand the committed myeloid progenitor compartments, rather than increase the rate of megakaryocyte maturation or the number of platelets released. We describe studies in which SCF stimulated murine CFU-Meg alone and in the presence of IL-3. However, a decrease in cultured cell plating density resulted in ablation of this SCF-stimulation of CFU-Meg colonies. CFU-Meg colony stimulation by SCF was dose dependent, even under serum-free conditions. The effects of SCF in other in vitro and in vivo animal model systems are reviewed.

Altered radioprotective properties of interleukin Iα (IL-1) in non-hematologic tumor-bearing animals

The radioprotective properties of IL-1 were investigated in the respective murine hosts for the Lewis lung (LLca) and EMT-6 tumors. For these studies, doses of total body irradiation were selected for the C57B1/6 (9.5 Gy) and Balb/c (7.5 Gy) mice that resulted in a 60% mortality over a 30-day interval. When a “priming” dose of 2.5 × 10 5 U IL-1 was administered 24 hr prior to the radiation exposure, animal mortality was markedly reduced (60% vs 5–10%). Under identical experimental conditions, however, the presence of either the LLca or the EMT-6 tumors in their respective host strains was found to compromise the level of radioprotection conferred by this priming dose of IL-1. In Balb/c mice bearing the EMT-6 tumor, a priming dose of IL-1 resulted in only a modest level of radioprotection when compared to non-tumor-bearing control animals (median animal survival increased by 11.5 days). In C57B1/6 mice bearing the LLca tumor, IL-1 failed to demonstrate any evidence of radioprotection. Following a sublethal dose of total body irradiation, the appearance of an accelerated repopulation of the stem cell (8d CFUs and CFU-GEMM) and the myeloid progenitor (CFU-M) compartments in the marrow of the IL-1 primed EMT-6, but not the LLca, tumor-bearing animals was consistent with the hypothesis that the mechanism leading to radioprotection in IL-I primed rodents involves an accelerated recovery of hematopoietic activity. It was also noted that the presence of the EMT-6 tumor was associated with an increase in the “radiosensitivity” of the Balb/c mouse. Collectively, these data suggest that the use of biological modifiers should be examined under altered physiological conditions prior to attempting to translate them into the clinic.

Cytogenetic Studies of Early Myeloid Progenitor Compartments in Ph -Positive Chronic Myeloid Leukemia. II. Long-Term Culture Reveals the Persistence of Ph -Negative Progenitors in Treated as Well as Newly Diagnosed Patients

Abstract We recently showed that long-term marrow cultures can be used to demonstrate the presence of Philadelphia (Ph1) negative progenitors in patients with newly diagnosed Ph1-positive chronic myeloid leukemia (CML). We now report results for 6 chronic phase patients studied 5–83 mo postdiagnosis and an additional 3 newly diagnosed patients. Marrow metaphases were exclusively Ph1-positive. Clonogenic assays revealed a minor population of Ph1-negative progenitors in 3 cases (1 treated, 2 untreated). Long-term marrow culture adherent layers contained Ph1- negative progenitors in 6 cases (3 treated, 3 untreated). Whenever this occurred, the Ph1-negative population had become the only one detectable within 3–4 wk, and this was always associated with a rapid decline of the Ph1-positive population. For 2 of the 3 cases where Ph1- negative progenitors were not detected, there was a similar rapid decline in the Ph1-positive population in culture. In the other case, Ph1-positive progenitors were maintained at levels typically seen in normal long-term marrow cultures. These results suggest that chromosomally normal stem cells may persist for a considerable period in the marrow of some, but perhaps not all, patients with CML, even in the face of maintenance chemotherapy. In addition, they provide new evidence of heterogeneity in this disease, as shown by the variable ability of Ph1-positive progenitor populations to be maintained in vitro.

Cytogenetic studies of early myeloid progenitor compartments in Ph1- positive chronic myeloid leukemia. II. Long-term culture reveals the persistence of Ph1-negative progenitors in treated as well as newly diagnosed patients

We recently showed that long-term marrow cultures can be used to demonstrate the presence of Philadelphia (Ph1) negative progenitors in patients with newly diagnosed Ph1-positive chronic myeloid leukemia (CML). We now report results for 6 chronic phase patients studied 5-83 mo postdiagnosis and an additional 3 newly diagnosed patients. Marrow metaphases were exclusively Ph1-positive. Clonogenic assays revealed a minor population of Ph1-negative progenitors in 3 cases (1 treated, 2 untreated). Long-term marrow culture adherent layers contained Ph1-negative progenitors in 6 cases (3 treated, 3 untreated). Whenever this occurred, the Ph1-negative population had become the only one detectable within 3-4 wk, and this was always associated with a rapid decline of the Ph1-positive population. For 2 of the 3 cases where Ph1-negative progenitors were not detected, there was a similar rapid decline in the Ph1-positive population in culture. In the other case, Ph1-positive progenitors were maintained at levels typically seen in normal long-term marrow cultures. These results suggest that chromosomally normal stem cells may persist for a considerable period in the marrow of some, but perhaps not all, patients with CML, even in the face of maintenance chemotherapy. In addition, they provide new evidence of heterogeneity in this disease, as shown by the variable ability of Ph1-positive progenitor populations to be maintained in vitro.

Cytogenetic studies of early myeloid progenitor compartments in Ph1-positive chronic myeloid leukaemia (CML). I. Persistence of Ph1-negative committed progenitors that are suppressed from differentiating in vivo.

We have cytogenetically analysed individual haemopoietic colonies to investigate the level and extent of normal stem cell suppression that occurs in patients with Philadelphia chromosome (Ph1)-positive CML. Seventeen patients were studied at diagnosis prior to the initiation of chemotherapy and five of these were studied again 1-16 months later. Another nine patients were studied for the first time 2-96 months after diagnosis and initiation of chemotherapy. No chromosomally normal metaphases were found in either direct marrow preparations or in haemopoietic colonies obtained from simultaneous assays of marrow and/or blood samples from 20 of the 26 patients studied. In the other six, chromosomally normal haemopoietic progenitors (BFU-E, CFU-C and CFU-G/E) were readily demonstrable even though in five of these patients all dividing cells in the bone marrow appeared to belong to the Ph1-positive clone at the time of study. These results indicate that the suppressive effects of clonal expansion on normal haemopoiesis are more pronounced, and apparent sooner, in the more differentiated compartments. In addition, they support the view that the original population of normal stem cells does not disappear rapidly, although their numbers may be diluted to undetectable levels depending upon the extent of clonal expansion at the stem cell level by the time of diagnosis.