Myeloid Progenitor Compartment Research Articles

Cohesin Subunit RAD21 Regulates the Differentiation and Self-Renewal of Hematopoietic Stem and Progenitor Cells.

Recent studies suggest that chromosomal cohesin complex proteins are important in regulating hematopoiesis and may contribute to myeloid malignancies. To investigate the effects of perturbing the cohesin subunit protein RAD21 on normal hematopoiesis, we used conditional knockout (cKO) mouse models. While cohesin is vital for hematopoietic stem cell (HSC) function, Rad21 haploinsufficiency (Rad21Δ/+) led to distinct hematopoietic phenotypes. Our findings revealed that Rad21Δ/+ cells exhibited decreased hematopoietic reconstitution in competitive bone marrow transplantation assays. This reduction in peripheral blood chimerism was specifically observed in the lymphoid compartment, while the chimerism in the myeloid compartment remained unaffected. Rad21 haploinsufficiency also resulted in changes in the hematopoietic stem and progenitor cells (HSPC) and myeloid progenitor compartments, with a significant accumulation of granulocyte-macrophage progenitors in the bone marrow. We observed differential gene expression in Rad21Δ/+ LSK (Lin- Sca1-Kit+) cells, including genes required for HSPC function and differentiation, such as Setdb1, Hmga2, Ncor1, and Myb. In addition, we observed a notable decrease in the expression of genes related to the interferon response and a significant reduction in the expression of genes involved in the IL2-STAT5 signaling pathways. Our studies suggest that RAD21 protein and level of its post-translational modifications in the bone marrow cells may play a potential role in hematopoiesis. Overall, Rad21 haploinsufficiency impairs hematopoietic differentiation and increases HSC self-renewal.

Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis.

In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different invivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 Tcells toward an activation state that includes IFN-γ production by these Tcell subsets invivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies.

Megakaryocyte progenitor cell function is enhanced upon aging despite the functional decline of aged hematopoietic stem cells.

SummaryAge-related morbidity is associated with a decline in hematopoietic stem cell (HSC) function, but the mechanisms of HSC aging remain unclear. We performed heterochronic HSC transplants followed by quantitative analysis of cell reconstitution. Although young HSCs outperformed old HSCs in young recipients, young HSCs unexpectedly failed to outcompete the old HSCs of aged recipients. Interestingly, despite substantial enrichment of megakaryocyte progenitors (MkPs) in old mice in situ and reported platelet (Plt) priming with age, transplanted old HSCs were deficient in reconstitution of all lineages, including MkPs and Plts. We therefore performed functional analysis of young and old MkPs. Surprisingly, old MkPs displayed unmistakably greater regenerative capacity compared with young MkPs. Transcriptome analysis revealed putative molecular regulators of old MkP expansion. Collectively, these data demonstrated that aging affects HSCs and megakaryopoiesis in fundamentally different ways: whereas old HSCs functionally decline, MkPs gain expansion capacity upon aging.

Single-Cell RNA Sequencing Reveals Distinct Hematopoietic Stem Cell Hierarchies in MDS

Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.

What Links Neutropenia to Immature Cardiolipin in Patients with Barth Syndrome (tafazzin-deficiency)?

Barth syndrome is an inherited X-linked disorder characterized by cardiomyopathy, skeletal muscle myopathy, and neutropenia. The syndrome arises because of inherited mutations in the gene TAZ, resulting in a loss of function of the protein tafazzin. Of note, a group of investigators recently described how tafazzin can regulate 'stemness' in models of acute myeloid leukemia (Cell Stem Cell, 2019). Tafazzin is an enzyme that processes the final step of cardiolipin maturation, replacing saturated with unsaturated acyl chains. Cardiolipin is a 4-tailed phospholipid that is almost-exclusively found in the inner membrane of the mitochondria. The lack of tafazzin activity results in a cardiolipin pool that contains more highly saturated lipid tails and it is this lack of unsaturated cardiolipins that contributes to a disorganized inner mitochondrial membrane. The link between tafazzin-deficiency and myopathy is generally explained by the dependence of muscle cells on mitochondrial function as well as oxidative respiration. The components of the electron transport chain are co-localized with cardiolipin in the inner mitochondrial membrane, and it is felt that their appropriate organization within the membrane lipid bilayer is dependent on the presence of mature cardiolipin which is lacking in those individuals with Barth syndrome. The link between tafazzin-deficiency and neutropenia is less clear. Neutrophils are terminally-differentiated effector cells of the innate immune system. They are critical for protection against bacterial and fungal pathogens and patients without sufficient neutrophils are among the most immunocompromised and at risk of lethal infection. Neutrophils have few mitochondria at baseline and are generally believed to rely primarily on glycolysis for energy production. It is not known if the mechanism of neutropenia in Barth syndrome is due to a lack of production or due to increased clearance (e.g. more prone to apoptosis). We undertook the study of tafazzin-deficient neutrophils to try to elucidate the mechanism of neutropenia in patients with Barth syndrome. We took advantage of an existing tafazzin-knockout mouse and a system of conditional immortalization of granulocyte-monocyte progenitors (GMP) using the ER-Hoxb8 system pioneered in our laboratory. This ER-Hoxb8 system allows for the unlimited ex vivo expansion of myeloid progenitors in the presence of estradiol and active Hoxb8. Once estradiol is removed from culture media, the Hoxb8 protein is inactive and the cells undergo normal, synchronous and terminal neutrophilic differentiation. In this manner, we were able to generate tafazzin-wild-type and knockout GMP lines from murine fetal liver cells. Analysis of the myeloid progenitor compartment in fetal liver cells (d14.5-d16.5) showed no difference between wild-type and knockout mice, arguing against a developmental defect (E15 results shown in PANEL A). Furthermore, the tafazzin-deficient ER-Hoxb8 GMPs and neutrophils were remarkably normal when tested across a variety of assays including phagocytosis, cytokine production and ROS generation (ROS by H2DCFDA shown in PANEL B). We hypothesized that the unpredictable neutropenia in patients with Barth Syndrome might be due to an increased proclivity to apoptosis because of the mitochondrial membrane defect. Indeed, the tafazzin-deficient GMPs showed an increased sensitivity to Bcl2-inhibition following treatment with ABT199 (PANEL C). Two lines of evidence have suggested that the increased tendency towards apoptosis may be due to endoplasmic-reticulum (ER) stress. (1) Transmission electron microscopy demonstrated 'swollen' ER in the tafazzin-deficient cells (not shown) and (2) a comparison of gene expression patterns demonstrated an increased expression of ATF4 and CHOP (DDIT3) in the tafazzin-deficient cells (PANEL D). We are now focused on validating these findings and in establishing models to confirm the ER-stress phenotype in vivo in the TAZ-knockout mouse model as well as primary samples from patients with Barth Syndrome. We hope that this line of work will confirm the mechanism of neutropenia and shed light on potential targets for therapeutic intervention. In addition, this very rare disorder has provided insight into a previously-unexpected link between neutrophil survival and the membrane integrity of the inner mitochondrial membrane. Figure Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder.

A novel prospective isolation of murine fetal liver progenitors to study in utero hematopoietic defects.

In recent years, highly detailed characterization of adult bone marrow (BM) myeloid progenitors has been achieved and, as a result, the impact of somatic defects on different hematopoietic lineage fate decisions can be precisely determined. Fetal liver (FL) hematopoietic progenitor cells (HPCs) are poorly characterized in comparison, potentially hindering the study of the impact of genetic alterations on midgestation hematopoiesis. Numerous disorders, for example infant acute leukemias, have in utero origins and their study would therefore benefit from the ability to isolate highly purified progenitor subsets. We previously demonstrated that a Runx1 distal promoter (P1)-GFP::proximal promoter (P2)-hCD4 dual-reporter mouse (Mus musculus) model can be used to identify adult BM progenitor subsets with distinct lineage preferences. In this study, we undertook the characterization of the expression of Runx1-P1-GFP and P2-hCD4 in FL. Expression of P2-hCD4 in the FL immunophenotypic Megakaryocyte-Erythroid Progenitor (MEP) and Common Myeloid Progenitor (CMP) compartments corresponded to increased granulocytic/monocytic/megakaryocytic and decreased erythroid specification. Moreover, Runx1-P2-hCD4 expression correlated with several endogenous cell surface markers’ expression, including CD31 and CD45, providing a new strategy for prospective identification of highly purified fetal myeloid progenitors in transgenic mouse models. We utilized this methodology to compare the impact of the deletion of either total RUNX1 or RUNX1C alone and to determine the fetal HPCs lineages most substantially affected. This new prospective identification of FL progenitors therefore raises the prospect of identifying the underlying gene networks responsible with greater precision than previously possible.

Stag2 regulates Hematopoietic Differentiation and Self-Renewal

Cohesin is a multimeric protein complex, which has been initially implicated in the cohesion of replicated sister chromatids but more recently shown to be involved in the long-range regulation of gene expression by stabilizing 3-dememsional structure of the genomic DNA. Recently, multiple components of the cohesin complex has been shown to be a recurrent target of somatic mutations in various myeloid malignancies, in which STAG2 is most frequently mutated and undergoes loss-of-function. However, the leukemogenic mechanism of mutated- STAG2 has not been fully understood, although recent studies have reported deregulated differentiation and enhanced self-renewal of STAG2 -mutated stem cells.To investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis, we generated Stag2 conditional knockout (cKO) mice with an Mx1 -cre allele, in which Stag2 deletion was induced by polyIC injection. When assessed in the peripheral blood of 12 to 20 week-old mice, the complete blood count showed no significant changes between Stag2 cKO mice and controls. No morphological alterations were observed in the peripheral blood as well as in the bone marrow. However, in the spleen, the extramedullary hematopoiesis was evident, exhibiting increased myeloid progenitors and erythroblasts. In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential in Stag2 deficient hematopoietic stem cells (HSCs) in vitro.Flow cytometry of bone marrow cells revealed increased numbers of hematopoietic stem and progenitor cells (HSPCs) defined as Lineage−Sca-1+Kit+(LSK) cells in Stag2 cKO mice compared with controls. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs), while decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs), compared to controls. Moreover, CD11b+Gr-1+ mature myeloid cells were significantly increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes myeloid skewing.In competitive bone marrow transplantation assays assessing the reconstitution potential, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. However, the reduced chimerism of Stag2 cKO-derived cells was largely confined to B-lymphocytes showing a severe reduction, while the chimerism of Stag2 cKO-derived myeloid cells was not affected compared to controls. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but rather tended to show increased numbers in the LSK, CMP and granulocyte/macrophage progenitor (GMP) fractions, while the MEP and CLP fractions were reduced. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs in vivo, but Stag2 -deleted cells may not uniformly contribute to all hematopoietic cell fractions probably due to the impaired differentiation of these HSCs.Next, we assessed the effect of Stag2 -deficiency on gene expression, where RNA sequencing of Stag2 -deleted HSPCs revealed a subset of genes differentially expressed between Stag2 WT and cKO cells. Including key myeloid-specific regulators, these genes were considered to be potential gene targets of Stag2, deregulation of which is implicated in the abnormal hematopoiesis of Stag2 cKO mice. Given that the cohesin complex is known to be involved in the establishing and maintaining DNA accessibility, we also assessed the global chromatin accessibility by assays for transposase accessible chromatin with sequencing (ATAC-seq) of Stag2 -deleted HSPCs. Of interest, chromatin accessibility in Stag2 -deleted cells was increased for genes implicated in myeloid differentiation, whereas reduced for those in lymphoid differentiation. Motif analysis of more accessible regions in Stag2 -deleted cells revealed an enrichment of the binding site for the transcription factor Runx1, which is known to regulate HSC differentiation.Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs through the modulation of chromatin accessibility and consequent abnormal gene expression, possibly contributing to leukemogenesis. DisclosuresTakaori-Kondo:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Research Funding; Janssen Pharma: Honoraria; Pfizer​: Honoraria.

Bone Marrow WT1 Levels in Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myelogenous Leukemia and Myelodysplasia: Clinically Relevant Time Points and 100 Copies Threshold Value

The outcome of allogeneic hematopoietic stem cell transplantation (HCT) in patients with myeloid malignancies is better in those without minimal residual disease (MRD) than in those with MRD+, as assessed by multiparametric flow cytometry (MPFC). WT1 quantitation also has been used to assess the probability of relapse in acute myelogenous leukemia (AML) treated with chemotherapy. We analyzed the clinical value of normalized bone marrow WT1 levels as a measure of the expanded myeloid progenitor compartment in a consecutive series of 193 adult patients with myeloid malignancies who underwent HCT. Bone marrow WT1 levels before the HCT, at the first bone marrow aspirate after infusion, and in the follow-up samples after HCT were determined by means of real-time PCR using the European LeukemiaNet normalized method. We sought to clarify the prognostic relevance in terms of overall survival (OS), progression-free survival (PFS), and cumulative incidence of relapse (CIR). Based on earlier experience in AML, we selected a threshold of 100 copies, defining 2 groups: patients with <100 WT1 copies and those with ≥100 copies. Patients with <100 WT1 copies before HCT (median time, 36 days; range, 4 to 268 days) had a better OS, PFS, and CIR than those with ≥100 copies (40 ± 1 versus 29 ± 6 days, P = .004; 35 ± 9 versus 26 ± 6 days, P = .002; and 29 ± 7 versus 37 ± 6 days, P = .051). In the first bone marrow study after the HCT (median time, 42 days; range 14 to 157 days, respectively), patients with <100 WT1 copies also had better outcomes in terms of OS, PFS, and CIR (40 ± 7 versus 31 ± 9 days, P = .025; 36 ± 7 versus 30 ± 8 days, P = .004; and 29 ± 6 days versus 54 ± 9, P < .001, respectively). At this time point, bone marrrow samples with >100 copies also included patients who were negative for MRD as assessed by MPFC (19 of 32). During the HCT follow-up, patients with sustained WT1 levels <100 copies showed a marked benefit in terms of OS, PFS, and CIR even compared with those with only a single measurement >100 copies (mean, 68 ± 11 versus 26 ± 7 days, P < .001; 63 ± 11 versus 20 ± 8 days, P < .001; and 20 ± 8 vs. 71 ± 8 days, P < .001, respectively). Standardized bone marrow WT1 levels using a 100-copy threshold in samples obtained before HCT, at leukocyte recovery, and during follow-up provided relevant prognostic information in patients with myeloid malignacies submitted to HCT.

An Altered Bone Marrow Vascular Niche Impacts Normal Hematopoiesis and Tilts the Balance Between Myelopoiesis and Lymphopoiesis

The bone marrow niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained to ensure their lifelong contribution to hematopoiesis. Recent evidence has highlighted the critical importance of the perivascular bone marrow (BM) niche as the key host and regulator of HSPCs. Bone marrow endothelial cells (BMECs) are major components of the vascular niche. While studies have shown that an alteration in a component of the niche can affect hematopoiesis and promote the development of myeloproliferative disorders/myelodysplastic syndromes, it remains unclear how altered BMECs can impact hematopoiesis. To this end, we have generated a Tamoxifen (TAM)-inducible Tie2-CreER/LSL-KRasG12D;tdTomato mouse model to introduce an oncogenic KRas mutation specifically in adult endothelial cells. The tdTomato reporter overlaps with the CD31 and vascular endothelial growth factor receptor 2 (VEGFR2) endothelial cell markers and shows no detectable leakage into the adult hematopoietic compartment. To evaluate changes in hematopoiesis, we performed complete blood counts at 12 weeks post TAM injection and found that the Tie2-CreER/LSL-KRasG12D mice (KRasG12D mice) had significantly more leukocytes (p=0.031) and neutrophils (p=0.002) than controls. Flow cytometry analysis confirmed that the KRasG12D mice had a significantly higher percentage of myeloid cells with concurrent decrease in lymphocyte percentage in the peripheral blood (p=0.016). At 16 weeks post TAM injection, a significant decrease in B cells could also be noted in the blood of KRasG12D mice (p=0.028). Compared to controls, the KRasG12D mice displayed splenomegaly (p=0.025) and their spleens had a higher percentage of myeloid cells (p=0.002). There was an increase in the common myeloid progenitor compartment in the spleen and a significant increase in the granulocyte macrophage progenitor compartment (p=0.014) of KRasG12D mice. These mice also had an increase in the short-term hematopoietic stem cell (ST-HSC) compartment both in the BM and spleen. Colony forming assays revealed that KRasG12D mice had a higher number of total colonies formed from BM (p=0.044), spleen (p=0.007) and blood cells (p=0.56). Genotyping PCR showed no KRasG12D activation in hematopoietic cells, confirming that the observed phenotypes were due to an effect in BMECs. To complement our native inducible mouse model, we transplanted BM cells from syngeneic BoyJ mice into lethally irradiated Tie2-CreER;KRasG12D or KRasWT recipients. The endothelial KRasG12D recipientsdied between 75-200 days post transplantation (p=0.0079) while the KRasWT recipients remained alive. The KRasG12D recipients also displayed splenomegaly (p=0.004). Competitive transplant studies with donor cells from KRasG12D or KRasWT mice with competitor cells from syngeneic mice (CD45.1) showed that BM cells from the KRasG12D mice (CD45.2) outcompeted cells from KRasWT mice with a significantly higher percentage of CD45.2 donor chimerism in all blood lineages examined. To uncover any molecular events underpinning these hematopoietic changes, we performed quantitative real-time polymerase chain reaction. Our preliminary experiments from total BM RNA of KRasG12D or KRasWT mice indicate that there is a significant increase in VEGFα and a decrease in transforming growth factor β in KRasG12D mice, accompanying the above noted increase in the ST-HSC population. Collectively, our data provide strong evidence that an abnormal vascular niche caused by oncogenic insults in BMECs can disrupt normal hematopoiesis and promote a myeloproliferative phenotype, thereby implicating abnormal BMECs as novel contributors to blood pathogenesis. Studies are underway to further assess the molecular contributions from the disrupted vascular niche and the resulting HSPCs. Uncovering the mechanism of how altered BMECs can remodel hematopoiesis holds the exciting promise of better therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

MiR-155 Promotes FLT3-ITD-Induced Myeloproliferative Disease through Inhibition of Interferon Signaling

Introduction & Background: MicroRNA expression is dysregulated in many human cancers, including hematologic malignancies. Among hematologic malignancies, acute myeloid leukemia (AML) carries a particularly poor prognosis, leading to over 10,000 deaths each year. The most common genetic aberration in AML is a gain-of-function mutation in the FMS-like tyrosine kinase 3 (FLT3) receptor. FLT3 internal tandem duplication (ITD) occurs in ~25% of all AML diagnoses, and confers a negative prognosis. MicroRNA expression has been shown to be dysregulated in FLT3-ITD+ AML, and miR-155 has been identified as the most highly overexpressed microRNA in this disease. MiR-155 is critical for the growth of FLT3-ITD+ cells in vitro, but the functional relevance of miR-155 in FLT3-ITD-mediated disease in vivo, and the downstream effects of miR-155 expression remain unclear.Methods and Design: In this study, we crossed mice homozygous for the FLT3-ITD mutation with miR-155 knockout mice to determine the specific role of miR-155 in the context of FLT3-ITD. The FLT3-ITD mice develop a chronic myeloid malignancy that we evaluated between 4-6 months of age, and compared this phenotype between groups (FLT3-ITD 155+/+ vs FLT3-ITD 155-/-).Results: FLT3-ITD miR-155-/- mice exhibited decreased myeloid expansion in the bone marrow, reduced splenomegaly, and decreased peripheral blood monocytosis and neutrophilia compared to their FLT3-ITD miR-155+/+ counterparts, indicating that miR-155 was playing a crucial role in promoting FLT3-ITD-mediated myeloproliferation. When examining the stem cell compartment of these animals, we found that miR-155 deficient animals had a reduced number of myeloid progenitors compared to their FLT3-ITD 155+/+ counterparts, suggesting miR-155 was critical for maintaining a robust myeloid progenitor pool in this disease. This phenotype was attributed to miR-155's role in promoting proliferation, but not survival, of the hematopoietic stem and progenitor cell (HSPC) and myeloid progenitor cell compartments in the bone marrow. RNA sequencing of the HSPC population in FLT3-ITD 155+/+ mice and FLT3-ITD 155-/- mice revealed that mice lacking miR-155 had an increased response to interferon, known to have a growth-suppressive effect on hematopoietic cells. We confirmed this finding through qPCR, where we found a number of interferon responsive genes were elevated in the HSPC and myeloid progenitor compartments of FLT3-ITD 155-/- mice compared to FLT3-ITD 155+/+ mice. We also observed increased protein levels of STAT1, a key transcription factor that activates interferon signaling, in the HSPCs and myeloid progenitors of FLT3-ITD 155-/- mice. These findings corresponded with human AML data from The Cancer Genome Atlas, where we found that FLT3-ITD+ AML samples had a decreased interferon signature compared to FLT3-WT AML samples. A number of putative miR-155 targets in the HSPCs are upregulated in the absence of miR-155, including PU.1, SHIP1, and CEBPB, all of which have previously been implicated as critical miR-155 targets in FLT3-ITD+ AML. Interestingly, CEBPB is known to regulate interferon responses.Conclusions and Future Directions: Our study establishes miR-155 as a critical promoter of FLT3-ITD-mediated myeloproliferative disease in vivo, a finding we attributed to miR-155's inhibition of the interferon response in this context. Further work will determine which of miR-155's targets could be potentiating this effect. We will also focus on the importance of miR-155 in FLT3-ITD+ leukemogenesis, as FLT3-ITD mice need collaborating mutations to undergo leukemic transformation. These findings suggest that miR-155 inhibitors may warrant clinical consideration as therapeutics in FLT3-ITD+ AML. DisclosuresNo relevant conflicts of interest to declare.

Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions

AbstractDisrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC–Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML.

Integrative Analysis of the Mutational Landscape of Mouse and Human AML Identifies Functionally Relevant Leukemia Disease Alleles

t(8;21) is the most frequent chromosomal abnormality in acute myeloid leukemia (AML), occurring in 4-12% of adult and 12-30% of pediatric patients. This translocation fuses the N-terminus of AML1 to nearly the entire coding region of ETO, resulting in expression of the fusion protein AML1-ETO. Observations that mice expressing AML1-ETO develop AML only if treated with mutagenic agents have suggested that AML1-ETO requires cooperating disease alleles for leukemogenesis. Consistent with this, t(8;21)+ AML patients harbor multiple genetic abnormalities. Recent exome/genome sequencing studies have expanded the number of known mutations in t(8;21)+ AML patients; however, efforts to distinguish driver from passenger mutations have yielded few cooperative events and the requirements for AML1-ETO leukemogenesis remain largely unknown.To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of two specific AML1-ETO driven mouse models of leukemia to the mutational profiles of human AML patients. We found that the mouse models of AML1-ETO driven AML were phenotypically similar in terms of their extensive latency, myeloid progenitor immunophenotype, and the acquired secondary disease alleles. The first model relies upon the expression of AML1-ETO in transplanted p21 null cells, while the second model relies upon the expression of AML1-ETO9a, a splice variant of AML1-ETO, in transplanted wild type cells.p21 is neither disrupted, nor methylated in t(8;21)+ AML. Because loss of p21 prevents the repair of damaged DNA, leukemogenesis may occur in this model once a cooperating disease allele has been naturally acquired in an AML1-ETO positive hematopoietic progenitor. AML1-ETO9a itself deregulates the expression of several DNA repair genes, suggesting that AML1-ETO9a could similarly facilitate the acquisition of a cooperating disease allele.When we compared the mutational landscape of these murine leukemias to AML patients, we found that the murine leukemias enrich for disease alleles present in human AML (hypergeometric p ≤ 4.26x10-20) and that there is a significant tendency for disease alleles mutated in both species to possess mutations in the same protein domain (hypergeometric p ≤ 4.23x10-3). Furthermore, domains mutated in both species were affected by recurrent mutations (Spearman correlation of domain p-values r = 0.53, p ≤ 2.73x10-8). While the frequency with which various protein classes were affected by mutations was significantly different in MLL-AF9 and AML1-ETO/AML1-ETO9a positive murine AML compared to MLL-fusion and t(8;21)+ positive human AML (p = 0.049), the protein classes targeted in AML1-ETO/AML1-ETO9a murine AML vs. human t(8;21)+ AML were not significantly different (p = 0.327).To identify disease alleles capable of cooperating with AML1-ETO, we determined that of the 424 genes mutated in both species, 38 of those genes were significantly mutated in human AML (Genome MuSiC SMG FDR ≤ 30%). These 38 genes represented 45 mouse orthologues, 38 of which were significantly mutated in AML1-ETO driven murine leukemias (FDR ≤ 10%). These 38 orthologues corresponded to 32 human orthologues, 3 of which were annotated in COSMIC as cancer-related genes: TET2, PTPN11, and THRAP3.Using retroviral transduction and transplantation experiments, we demonstrated that the expression of AML1-ETO in transplanted Tet2 null cells or PTPN11 D61Y cells was sufficient for leukemogenesis. At euthanasia, mice exhibited leukocytosis, anemia, thrombocytopenia, splenomegaly, and an expansion in the myeloid progenitor compartment. Our identification of Tet2 loss as a cooperating allele implicates mutations in epigenetic regulators as potential driving events in t(8;21)+ AML, while the discovery of PTPN11 D61Y solidifies the role of constitutive MAPK signaling in t(8;21)+ AML.This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)+ AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML. Collectively, these findings illustrate the power of integrating murine and human genomic profiling to identify functionally relevant disease alleles in AML. DisclosuresLevine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

The Hedgehog Regulated Transcription Factor and Tumor Suppressor, Gli3, Regulates Hematopoietic Stem Cell Number, Self-Renewal and Differentiation

The Hedgehog (Hh) signaling pathway plays a critical role in embryonic development and adult tissue homeostasis and has emerged as an important therapeutic target in many cancers, including leukemia and myeloproliferative diseases. Our mechanistic understanding of Hh pathway signaling and regulation comes primarily from developmental studies in neural and limb development. Studies of Hedgehog signaling in the hematopoietic system have produced contradictory results, and no clear consensus regarding Hh signaling in normal hematopoiesis is available to inform the role of Hedgehog signaling in hematologic malignancies. In our work we have focused on understanding the downstream effectors of Hedgehog signaling, the Gli transcription factors. The three Gli proteins, Gli1, Gli2 and Gli3 have both transcriptional activator and repressor functions, which allow for regulation and fine-tuning of Hedgehog pathway output. Previous studies from our group have revealed that Gli1null HSCs had no defects in self-renewal, however myeloid differentiation and stress hematopoiesis were severely impaired (Merchant, et al., Blood 2010). In normal tissues, Hh pathway activation via Ptch/Smo causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor Gli3R. Our recent studies demonstrated that GLI3R has a tumor suppressor role in human acute myeloid leukemia by directly repressing AKT expression (Chaudhry et al., AACR Annual Meeting 2015). To date nothing is known about the role of Gli3 in normal hematopoiesis. In the present study, we crossed Vav-Cre transgenic mice to Gli3fl/fl mice to generate mice with a conditional loss of Gli3 (Gli3null) in the hematopoietic system. HSC self-renewal was analyzed by serial transplant. In comparison to HSCs from Gli3 wild type (Gli3WT) mice bone marrow (BM), HSCs from Gli3null BM showed decreased long-term engraftment and self-renewal. In addition, quantification of long-term HSC (LT-HSC, CD34neg Flt3neg KSL), short-term HSC (ST-HSC, CD34+ Flt3neg KSL), and multi-potent progenitor (MPP, CD34+ Flt3+ KSL) revealed that the frequency of LT-HSCs in Gli3null BM (0.004-0.007%) was lower compared to Gli3 WT BM (0.008-0.02%). In mice transplanted with Gli3null BM, myeloid expansion was observed with a block in T and B cell lineage differentiation. Analysis of the c-Kit+ Sca1neg Linneg (KL) myeloid progenitor compartment revealed a two-fold increase in the FcRγhigh CD34+ KL granulocyte-monocyte progenitors (GMPs) in Gli3null BM, suggesting an expansion of granulocytic compartment. Since Gli3R is a key negative regulator of Gli1, these are consistent with decrease in GMP and myeloid differentiation previously seen in Gli1null mice. In summary, our studies reveal a previously unknown function for Gli3 in regulating HSCs and myeloid differentiation, and help to elucidate the complex regulation of Hh signaling in the hematopoietic system. DisclosuresNo relevant conflicts of interest to declare.

C/EBPβ-Mediated Expansion of Hematopoietic Stem/Progenitors Precedes ‘Emergency' Granulopoiesis Induced by Candidemia.

Abstract 2336Granulopoiesis, the process of granulocyte production in the bone marrow (BM), is tightly regulated to meet host demands during both ’steady state’ and ’emergency’ situations such as infections. The transcription factor CCAAT/Enhancer Binding Protein β (C/EBPβ) plays critical roles in emergency granulopoiesis (Hirai et al. Nat Immunology, 2006). However, the precise developmental stages in which C/EBPβ is required are unknown. In this study, we investigated the roles of C/EBPβ in the proliferation and differentiation of prospectively identified intermediates between hematopoietic stem cells and mature granulocytes in mouse BM.In order to analyze the mouse BM cells undergoing granulopoiesis, novel flow cytometric method was developed. Mouse BM cells retaining the ability to give rise to granulocytes were dissected into five distinct subpopulations (#1–#5) according to their levels of c-kit and Ly-6G expression. Upon infection of Candida albicans (4 × 106 CFU/20 g body weight/mouse) on day 1, C/EBPβ was upregulated at the protein level but not at mRNA level in all the granulopoietic subpopulations, suggesting the importance of the transcription factor in □emergency’ granulopoiesis.Then, the role of C/EBPβ was further assessed by analyzing C/EBPβ knockout (KO) mice. At steady state, the distribution of granulopoietic cells in BM of C/EBPβ KO mice at □esteady state’ was identical to that of wild type (WT) mice. In contrast, the rapid increase in immature subpopulations #1 and #2 observed in WT mice at 1 day post-infection was significantly attenuated in C/EBPβ KO mice. The levels of mRNA expression for granule proteins (cathepsin G, myeloperoxidase, elastase 2, proteinase 3, lactoferrin and MMP9) within each subpopulation from WT and C/EBPβ KO mice were identical at both steady state and during infection. When the cell cycle status of these models was evaluated using in vivo BrdU labeling experiments, incorporation of BrdU in subpopulation #1 and #2 in C/EBPβ KO mice was always slightly lower than in WT mice, but the differences were not statistically significant. These findings suggest that C/EBPβ is required for efficient proliferation of early granulocytic precursors but not directly involved in the differentiation/maturation process.To elucidate the roles of C/EBPβ in the proliferation of the early granulopoietic subpopulations, the hematopoietic stem cells (HSCs) and myeloid progenitor compartments were analyzed in WT and C/EBPβ KO mice. The frequency and number of c-kit+ Sca-1+ lineage markers− HSC were identical between WT mice and C/EBPβ KO mice during the steady state, and were not significantly affected on day 1 post-infection. Induction of candidemia increased the frequency and number of granulocyte-macrophage progenitors (GMP) in WT mice, and these increases were significantly attenuated in C/EBPβ KO mice. Upon induction of candidemia, the frequency of BrdU-positive cells in the HSC and common myeloid progenitors (CMP) populations from WT mice increased significantly; however, an increase of BrdU-positive cells was observed only within the HSC compartment in C/EBPβ KO mice, and at a lower level than that in WT miceTaken together, these data suggest that the proliferation of early granulocytic precursors is tightly coupled to differentiation/maturation and that C/EBPβ is involved in the efficient amplification of early granulocyte precursors including HSC and myeloid progenitors during candidemia-induced ’emergency’ granulopoiesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression and Function of PML-RARA in the Hematopoietic Progenitor Cells of Ctsg-PML-RARA Mice

Because PML-RARA-induced acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, many groups have speculated about whether its leukemic cell of origin is a committed myeloid precursor (e.g. a promyelocyte) versus an hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg, and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice), are expressed in the purified KLS cells of these mice (KLS = Kit+Lin−Sca+, which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Further, we demonstrate the transcriptional consequences of PML-RARA expression in Ctsg-PML-RARA mice in early myeloid development in other myeloid progenitor compartments [common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs)], which have a distinct gene expression signature compared to wild-type (WT) mice. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice and alters the transcriptional signature of these cells, it does not induce their self-renewal. In sum, these results demonstrate that in the Ctsg-PML-RARA mouse model of APL, PML-RARA is expressed in and affects the function of multipotent progenitor cells. Finally, since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in this mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.

TLR-Signaling Induces Granulocytic MDSCs Generation by Directing Myeloid Lineage Commitment During Polymicrobial Sepsis

Abstract 2160 Introduction:Elevated leukocyte frequency at early stage of polymicrobial sepsis is a common phenomenon, which is considered to be a result of rapid migration of neutrophils from the bone marrow (BM). However, a dramatic increase of granulocytic myeloid-derived suppressor cells (MDSCs) of sepsis usually occurs after sustained stimuli of the pathogens, which implies that emergency granulopoiesis is involved in negative regulation of immune response. In the present study, we try to reveal the mechanisms by which TLR signal regulates emergency myelopoiesis during infection. Methods and models:B Mice were subjected to cecal ligation and puncture to make polymicrobial sepsis model. Blood, spleens and bone marrow were harvested post operation at 1, 3, 7 and 12 day, and analyzed using flow cytometry. Hematopoietic stem cells and special progenitors were isolated using an FACS Aria sorter, and then total mRNA was extracted and reverse-transcripted. Some lineage commitment-related genes including Ikzf1,Gfi1, Cebp/a, PU.1, Csf1r, Csf2r, Csf3r, Notch1, IL-7r, Flt3 were analyzed by Real-time PCR. The methylcellulose colony assay of single-cell suspensions of bone marrow cells was used to identify myeloid progenitors in sepsis model. Results:We found that the elevated frequency of myeloid cells was associated with an expansion of hematopoietic stem cell (HSC) pool accompanied by augmented proportion of cells entering cell cycle. Furthermore, increased HSC numbers resulted in gradually elevated proportions of common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs), but a decrease of the frequency of common lymphoid progenitors (CLPs). In line with the decreased ratio of lymphoid/myeloid progenitors, real-time PCR revealed impaired expression levels of lymphoid differentiation-related genes in HSCs and CLPs, including Flt3, IL7R, Notch1, Ikaros and Rag1. By contrast, the expression levels of myeloid differentiation-related genes (eg., CEBP/a, Gfi1, PU.1, GM-CSFR) were up-regulated or remained unchanged in the stem cell and myeloid progenitor compartments. Moreover, consistent with elevated frequency of granulocytic MDSCs, an increase of Gfi-1 expression which made a granulocytic decision and a decrease of M-CSFR expression which played a crucial role in monopoiesis were noticed in CMPs and GMPs. More importantly, similar results were obtained in the mice challenged with LPS peritoneal injection. Conlusion:Our study sheds light on its importance of TLR-signaling in generation and replenishment of granulocytic MDSCs by affecting early myeloid /lymphoid lineage decision and late granulocyte /monocyte lineage decision. Disclosures:No relevant conflicts of interest to declare.

Mir-590-5p, Mir-219-5p, Mir-15b and Mir-628-5p Are Commonly Regulated by IL-3, GM-CSF and G-CSF in Acute Myeloid Leukemia,

Abstract 3520IL-3, GM-CSF and G-CSF are predominant regulators for growth and differentiation of myeloid progenitors. Interestingly, they all signal via a common JAK2-STAT5 pathway in myeloid progenitor compartments. However, the specific mechanism through which JAK2-STAT5 responds differentially to early-acting and lineage restricted cytokines, particularly in leukemic and stem/progenitor cells, is largely unresolved. Aberrations in IL-3, GM-CSF and G-CSF induced signaling are frequently reported in acute myeloid leukemia (AML). microRNA (miRNA) play several crucial roles during hematopoiesis that include lineage decisions, stem cell progenitor transitions, niche control and other cell functions. Recent investigations have linked aberrant miRNA expression with AML. We hypothesized that a unique response of leukemic myeloid progenitors to IL-3, GM-CSF, and G-CSF are possibly mediated in part by distinct regulation at the miRNA level. Therefore, herein, by utilizing a unique leukemic myeloid cell line, AML-193, that responds to both early and late acting cytokines, we profiled IL-3, GM-CSF and G-CSF regulated miRNA signatures in leukemic myeloid progenitors. For miRNA profiling, AML-193 cells were initially exposed to IL-3 for 3 days followed by GM-CSF for 3 more days and subsequently to G-CSF for 3 days. We then profiled miRNA expression induced by IL-3, GM-CSF and G-CSF in AML-193 cells by treating the respective cohorts post growth factor deprivation with corresponding cytokines. Using SA Bioscience’s complete Human V2.0 miRNA Genome Array platform for real-time qPCR-based miRNA expression profiling, we investigated miRNA signatures regulated by IL-3, GM-CSF and G-CSF for n=704 miRNAs in AML-193 cells. Frequencies of false positives were avoided using stringent filters. IL-3 specifically regulated 54 miRNAs and those miRNAs that were highly regulated included miR-362-3p, miR-590-3p, miR-340, miR-24-2, miR-1183 and miR-99a. GM-CSF specifically regulated miRNAs included let-7f, let-7a*, miR-195, miR-122, miR-376-c and miR-33a. G-CSF specifically regulated set included miR-21*, miR-192*, miR-32*, miR-7-1*, miR-545* and miR-37-4a*. Interestingly, 301 miRNAs were commonly regulated by IL-3, GM-CSF and G-CSF. Among the commonly regulated miRNAs, the ones that were subjected to high levels of regulation included miR-590-5p, miR-219-5p, miR-92-a1*, miR-378*, miR-548-3p, miR-29a*, miR-590-3p, miR-203, miR-363, miR-454, miR-340, miR-196a, miR-152, miR-10b, miR-24-2*, miR-10a, miR-182, miR-27a*, and miR-199a-3p. Interestingly, the commonly regulated miRNAs demonstrated a directional regulation in the order of IL-3>GM-CF>G-CSF. Analysis of the potential targets of significantly regulated miRNAs revealed important functional roles in myeloid cell development and differentiation. Conserved targets of miR-590-5p included BMPR2, PCBP2 and KLF3. Targets for miR-219-5p included Smad4 and GADD45b. Targets for miR-362-3p included Sox17, an inhibitor of the Wnt signaling pathway. Furthermore, to identify the JAK2-STAT5 responsiveness of miR-219-5p, miR-362-3p, and miR-590-5p, a STAT5 specific inhibitor (N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide) was used prior to cytokine treatment. Interestingly, IL-3 and GM-CSF mediated induction of miR-590-5p, miR-219-5p and miR-362-3p were not significantly affected by STAT5 inhibitor. However, the G-CSF induction of miR-219-5p and miR-590-5p were significantly inhibited by the STAT5 specific inhibitor. In summary, we have discovered for the first time novel miRNA profiles regulated by IL-3, GM-CSF and G-CSF in an acute myeloid leukemia progenitor cell model. Importantly, we have established a cytokine regulated miRNome for leukemic myeloid progenitors and set the stage for future investigations in leukemic stem cells to delineate the pathological roles of dysregulated miRNAs in AML. In addition, our study displayed that G-CSF induced a subset of miRNAs that are principally regulated via the STAT5 circuit in AML. Further, these novel miRNA signatures may have therapeutic implications for targeting dysregulated miRNAs by antagomir strategy or miRNA replacement therapy, paving the way for the development of novel miRNA-based therapeutic interventions in AML. Disclosures:No relevant conflicts of interest to declare.

A high omega-3 fatty acid diet has different effects on early and late stage myeloid progenitors.

The effects of the polyunsaturated omega-3 (n-3) and omega-6 (n-6) fatty acids (FA) on hematopoiesis are complex in that both FA forms are processed into leukotrienes, eicosanoids, and prostaglandins, which can have independent effects. These FA have antagonistic effects in that n-6 FA prostaglandins tend to be pro-proliferative and pro-inflammatory, while the effects of n-3 FA prostaglandins are the opposite. We have previously shown that diets high in n-3 FA reduce the size of the middle to later stage myeloid progenitor compartment in FVB X sv129 F(1)hybrid mice. To assay the effects of high n-3 FA diets on earlier stages of myelopoiesis, we fed C57BL/6J mice diets high in n-3 FA or levels of n-3/n-6 FA similar to western diets and assayed the effects on myelopoiesis with flow cytometry and colony forming cell assays. Results indicate an expansion of the common myeloid progenitor cell compartment in high n-3 FA diets, which does not persist into later stages where the number of progenitor cells is actually lower in high n-3 FA fed animals. Investigations in vitro with the hematopoietic stem cell line EML-clone 1 indicate that cells cultured with eicosapentaenoic acid (n-3 FA) or arachidonic acid (n-6 FA) have no differences in cell viability but that arachidonic acid more rapidly produces progenitors with low levels of the macrophage developmental marker, F4/80.

The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome

AbstractThe pseudo tyrosine kinase receptor 7 (PTK7) is an orphan tyrosine kinase receptor assigned to the planar cell polarity pathway. It plays a major role during embryogenesis and epithelial tissue organization. Here we found that PTK7 is also expressed in normal myeloid progenitors and CD34+ CD38− bone marrow cells in humans. We performed an immunophenotyping screen on more than 300 patients treated for hematologic malignancies. We demonstrated that PTK7 is expressed in acute myeloid leukemia (AML) and is mostly assigned to granulocytic lineage differentiation. Patients with PTK7-positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced leukemia-free survival in a multivariate analysis model. In vitro, expression of PTK7 in cultured leukemia cells promotes cell migration, cell survival, and resistance to anthracycline-induced apoptosis. The intracellular region of PTK7 is required for these effects. Furthermore, we efficiently sensitized primary AML blasts to anthracycline-mediated cell death using a recombinant soluble PTK7-Fc protein. We conclude that PTK7 is a planar cell polarity component expressed in the myeloid progenitor compartment that conveys promigratory and antiapoptotic signals into the cell and that represents an independent prognosis factor of survival in patients treated with induction chemotherapy.

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

BackgroundSignal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. However, the manner in which STAT5 responds to early-acting and lineage-restricted cytokines, particularly in leukemic stem/progenitor cells, is largely unknown.Methodology/Principal FindingsWe optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at a 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. 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.Conclusions/SignificanceIn conclusion, our optimized multiparametric flow cytometry protocols allow the analysis of signal transduction at the single cell level in normal and leukemic stem and progenitor cells. Our study demonstrates highly distinctive cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.