ALBERT

Description: To explore whether and how T cells can affect myelopoiesis, we investigated myeloid differentiation in a model for T cell-mediated immune activation. We found that CD70-transgenic (CD70TG) mice, which have elevated numbers of interferon-γ (IFN-γ)–producing effector T cells in the periphery and bone marrow, are almost devoid of eosinophilic granulocytes. Induction of allergic airway inflammation in these mice failed to induce eosinophilia as well as airway hyperresponsiveness. CD70TG mice also have strongly reduced numbers of eosinophil lineage-committed progenitors, whereas granulocyte/macrophage progenitors from these mice are unable to generate eosinophils in vitro. We found that granulocyte/macrophage progenitors express IFN-γR1 and that IFN-γ is sufficient to inhibit eosinophil differentiation of both murine and human progenitor cells in vitro. We demonstrate that inhibition of eosinophil development in CD70TG mice is IFN-γ–dependent and that T cell–derived IFN-γ is sufficient to inhibit eosinophil formation in vivo. Finally, we found that IFN-γ produced on anti-CD40 treatment and during viral infection can also suppress eosinophil formation in wild-type mice. These data demonstrate that IFN-γ inhibits the differentiation of myeloid progenitors to eosinophils, indicating that the adaptive immune system plays an important role in orchestrating the formation of the appropriate type of myeloid cells during immune activation.

Description: Limited number of hematopoietic stem cells in umbilical cord blood (UCB) presents a problem when using UCB for stem cell transplantation. Improving their homing capacity could reduce the need for high initial cell numbers during transplantation procedures. Although it is evident that protein kinase B (PKB/c-Akt) plays an important role in regulation of migration of various cell types, a role for PKB in regulation of migration and homing of human hematopoietic stem and progenitor cells remains to be determined. PKB activity was found to be required for induction of adhesion to bone marrow–derived stromal cells and detrimental for migration of UCB-derived CD34+ hematopoietic progenitors. In addition, PKB activity was found to positively regulate integrin expression. CD34+ hematopoietic progenitors, and their capacity to form colonies in vitro, were not affected by transient inhibition of PKB. Finally, transplantation of β2-microglobulin−/− nonobese diabetic/severe combined immunodeficient mice with CD34+ cells ectopically expressing constitutively active PKB resulted in reduced migration to the bone marrow, whereas inhibition of PKB activity resulted in an induction in bone marrow homing and engraftment. These results indicate that transient inhibition of PKB activity may provide a means for ex vivo stem cell manipulation to improve bone marrow transplantation regimes.

Description: Background and Objectives Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognosis associated with ETV6-RUNX1 positive ALL, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. Leukemic cells reside in the bone marrow microenvironment, where they are nurtured and protected against chemotherapy. In this study, we investigated novel ways to disrupt this leukemic niche by targeting signaling pathways contributing to the migration of ETV6-RUNX1 positive leukemic cells. Results Gene expression profiling and subsequent pathway analysis of leukemic blasts of 654 ALL patients revealed a significant enrichment of genes involved in regulation of cellular movement and cell morphology in ETV6-RUNX1 positive BCP-ALL patients compared with ETV6-RUNX1 negative BCP-ALL patients (p 〈 0.001). In correspondence, the same pathways were significantly upregulated in cord blood-derived hematopoietic progenitor cells (CB-CD34+) ectopically expressing ETV6-RUNX1 (p 〈 10E-06). LARG (ARHGEF12) was identified to be the most important regulator of this pro-migratory signature. This gene encodes for the G-protein-regulated Rho Guanine Exchange Factor 12, a specific activator of the GTPase RhoA. LARG expression was 5.7-fold higher in ETV6-RUNX1 positive BCP-ALL cells than in ETV6-RUNX1 negative BCP-ALL cells (p 〈 10E-06). Similarly, LARG was upregulated 5.4-fold in CB-CD34+ cells expressing ETV6-RUNX1 compared with empty vector controls (p = 0.03). To determine the importance of the LARG/RhoA pathway in the induction of this migratory phenotype, we used two recently identified small molecule inhibitors of the LARG/RhoA pathway: Y16, a specific LARG inhibitor, and G04 (Rhosin), a specific RhoA inhibitor. Both inhibitors significantly reduced the migration of ETV6-RUNX1 positive leukemic cells towards a gradient of CXCL12 (75%, p 〈 0.01) whereas the migration potential of ETV6-RUNX1 negative leukemic cells remained unaffected. Migration of ETV6-RUNX1 positive leukemic cells towards patient derived mesenchymal stromal cells was inhibited to a similar extent (75-85%, p 〈 0.01). In contrast, LARG/RhoA inhibition in ETV6-RUNX1 negative cells resulted in an induction of migration towards MSCs. While LARG/RhoA inhibitors reduced migration of ETV6-RUNX1 positive cells in a targeted manner, inhibition of CXCR4 by the CXCR4 antagonist AMD3100 (Plerixafor) reduced the migration of both ETV6-RUNX1 positive cells and ETV6-RUNX1 negative BCP-ALL cells. Next, we studied the additional cellular effects of LARG/RhoA inhibition on leukemic cells. shRNA-mediated silencing of LARG modestly reduced the proliferation rate of both ETV6-RUNX1 positive and ETV6-RUNX1 negative cell lines (p

Description: Background: Translocation t(12;21), resulting in the ETV6-RUNX1 fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite the favorable prognostic parameters of this B-ALL subgroup, relapse and resistance to chemotherapeutics occur and treatment-induced side effects are considerable. The molecular mechanisms underlying ETV6-RUNX1-driven leukemia are largely unknown. Increased knowledge of these mechanisms is essential to develop novel therapeutic strategies to selectively target ETV6-RUNX1-positive leukemia. Objectives: This study aims to identify and target the molecular drivers behind ETV6-RUNX1-positive BCP-ALL. Results: Gene expression profiling of leukemic blasts of 654 ALL patients revealed that the class III PI3-kinase Vps34, an important regulator of autophagy, was exclusively up-regulated in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (2.7-fold; p ≤ 10-30). In addition, ectopic expression of ETV6-RUNX1 in cord blood-derived hematopoietic progenitor cells (CB-HPCs) significantly induced expression of Vps34 1.3-fold already 40 hours after transduction (p ≤ 0.05). This suggests that the Vps34-autophagy pathway is activated by ETV6-RUNX1, which may mechanistically explain the leukemogenic and pro-survival properties ascribed to ETV6-RUNX1. In correspondence, Ingenuity Pathway Analysis (IPA) predicted a pro-survival and pro-proliferative phenotype in ETV6-RUNX1 transduced CB-HPCs and highlighted a network of up-regulated transcription factors, including HEY1, EGR1, GATA1 and GATA2 (2 – 25-fold up-regulation; p ≤ 0.05). Luciferase reporter assays revealed that not only the ETV6-RUNX1 fusion protein, but also the ETV6-RUNX1-induced target genes HEY1, EGR1 and GATA1 positively regulate Vps34 promoter activity (5 – 13-fold up-regulation; p ≤ 0.01).Lentiviral knockdown experiments were performed to elucidate the importance of Vps34 expression in ETV6-RUNX1-positive BCP-ALL cells. Knockdown of all Vps34 transcript variants, with two independent constructs, led to complete growth arrest of the ETV6-RUNX1-positive cell lines REH and AT2, while this only led to a decrease in proliferation of the ETV6-RUNX1-negative cell line NALM6. This growth arrest was caused by a significant induction of apoptosis (more than 4-fold 7 days after transduction; p ≤ 0.001) and a significantly reduced percentage of cycling cells (1.3-fold 7 days after transduction; p ≤ 0.05). Analysis of p62 protein expression by western blot and reverse phase protein arrays revealed that the levels of autophagy were significantly higher in ETV6-RUNX1-positive compared to ETV6-RUNX1-negative BCP-ALL patients (p ≤ 0.001). In addition, knockdown of ETV6-RUNX1 and Vps34 significantly reduced autophagy, quantified with confocal microscopy, in ETV6-RUNX1-positive cells with 50% and 84%, respectively (p ≤ 0.01). Furthermore, pharmacological inhibition of autophagy with hydroxychloroquine (HCQ) significantly reduced cell viability of BCP-ALL cell lines and primary patient-derived BCP-ALL cells (p ≤ 0.001). Treatment of the ETV6-RUNX1-positive BCP-ALL cell lines REH and AT2 with 20 µg/mL HCQ resulted in a 82% and 95% reduced cell viability, while the viability of ETV6-RUNX1-negative BCP-ALL cell lines and T-ALL cell lines were reduced to a lesser extent (NALM6: 43%; TOM-1: 50%; Loucy: 40%; Jurkat: 0%). Importantly, HCQ selectively sensitized ETV6-RUNX1-positive leukemic cells to L-asparaginase treatment in clinically relevant concentrations. Treatment of primary ETV6-RUNX1-positive patient cells with 10 µg/mL HCQ resulted in a 70% reduction in cell survival during L-asparaginase exposure (p ≤ 0.01). This sensitization was not observed in ETV6-RUNX1-negative BCP-ALL cells. Conclusion: The ETV6-RUNX1 fusion protein activates autophagy via Vps34, which is essential for survival and proliferation of ETV6-RUNX1-positive cells. Inhibition of autophagy in primary ETV6-RUNX1-positive leukemic cells inhibited cell survival and sensitized these cells to L-asparaginase treatment. These results indicate that autophagy inhibition may provide a novel means to sensitize L-asparaginase-resistant ETV6-RUNX1-positive BCP-ALL patients. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoiesis is a highly regulated process resulting in the formation of all blood lineages. Aberrant regulation of phosphatidylinositol-3-kinase (PI3K) signaling has been observed in hematopoietic malignancies, suggesting that regulated PI3K signaling is critical for regulation of blood cell production. An ex vivo differentiation system was used to investigate the role of PI3K and its downstream effector, protein kinase B (PKB/c-akt) in myelopoiesis. PI3K activity was essential for hematopoietic progenitor survival. High PKB activity was found to promote neutrophil and monocyte development, while, conversely, reduction of PKB activity was required to induce optimal eosinophil differentiation. In addition, transplantation of β2-microglobulin (−/−) NOD/SCID mice with CD34+ cells ectopically expressing constitutively active PKB resulted in enhanced neutrophil and monocyte development, whereas ectopic expression of dominant-negative PKB induced eosinophil development in vivo. Inhibitory phosphorylation of C/EBPα on Thr222/226 was abrogated upon PKB activation in hematopoietic progenitors. Ectopic expression of a nonphosphorylatable C/EBPα mutant inhibited eosinophil differentiation ex vivo, whereas neutrophil development was induced, demonstrating the importance of PKB-mediated C/EBPα phosphorylation in regulation of granulopoiesis. These results identify an important novel role for PKB in regulation of cell fate choices during hematopoietic lineage commitment.

Description: An important mediator of cytokine signaling implicated in regulation of hematopoiesis is the PI3K/protein kinase B (PKB/c-Akt) signaling module. Constitutive activation of this signaling module has been observed in a large group of leukemias. Because activation of this signaling pathway has been demonstrated to be sufficient to induce hematologic malignancies and is thought to correlate with poor prognosis and enhanced drug resistance, it is considered to be a promising target for therapy. A high number of pharmacologic inhibitors directed against either individual or multiple components of this pathway have already been developed to improve therapy. In this review, the safety and efficacy of both single and dual-specificity inhibitors will be discussed as well as the potential of combination therapy with either inhibitors directed against other signal transduction molecules or classic chemotherapy.

Description: Epigenetic modifications play an important role in the pathogenesis of myeloid malignancies of which alterations in cellular acetylation profiles have proven to contribute substantially. The clinical use of chromatin modulating drugs, such as histone deacetylase (HDAC) inhibitors, has more recently been evaluated in several clinical trials. Nonetheless, little is known concerning the effects of HDAC inhibitors on myelopoiesis or their specific targets in human hematopoietic stem cells. In order to investigate this, we utilized a differentiation system in which umbilical cord blood derived CD34+ cells were differentiated ex-vivo in liquid and semi-solid cultures. We investigated the effect on lineage development, colony-forming potential, proliferation and differentiation using trichostatin A (TSA; group I/II/IV, 5–25nM), sodium butyrate (SB; group I/IIa, 100–500μM) and valproic acid (VPA; group I/IIa, 100–500μM). In our liquid culture system we observed that TSA increased the numbers of CD34+ hematopoietic progenitors resulting in a dose dependent delay in granulocyte/macrophage progenitor (GMP) differentiation. Furthermore, TSA promoted differentiation towards megakaryocyte/erythroid progenitors (MEP) in the absence of thrombopoietin (TPO) and erythropoietin (EPO). SB promoted a shift towards the granulocyte/macrophage lineage, and inhibited proliferation and differentiation of CD34+ progenitors. VPA increased the number of CD34+ hematopoietic progenitors in a dose dependent manner, with increased numbers of MEP and a delay in GMP differentiation. To investigate the colony-forming potential of hematopoietic progenitors and further define the effect on lineage choice decisions in the presence of HDAC inhibitors, we also used a semi-solid culture system. In agreement with the results of the progenitor analysis, 100–200mM VPA stimulated granulocyte/macrophage colony formation which was also observed after 100mM SB treatment, although colonies were smaller and less differentiated. All three HDAC inhibitors showed a dose dependent inhibition of erythroid colony-forming potential and differentiation. We also used the liquid culture system to examine proliferation during neutrophil development. TSA moderately increased CD34+ progenitor expansion in a dose dependent manner, while 100–500mM SB and 500mM VPA abrogated expansion, eventually leading to decreased cell survival. In contrast, 100–200mM VPA increased cell expansion without affecting differentiation potential. Neutrophil differentiation was improved, although not significantly, by TSA, while SB inhibited neutrophil differentiation showing dysplastic features and signs of early apoptosis. Treatment with 500μM VPA also inhibited neutrophil differentiation reflected by an increased amount of common myeloid progenitors (CMP), a differentiation block in immature neutrophils and decreased cell survival. In order to determine the effects of HDAC inhibition on histone acetylation we performed Western Blot analysis on protein lysates from hematopoietic progenitors. SB and VPA strongly increased acetylation of total H4 with hyperacetylation of H4K16 and this effect was observed to a lesser extent with TSA. During myeloid differentiation, all three HDAC inhibitors clearly increased total H3 acetylation as represented by H3K9 hyperacetylation. Taken together, we observed comparable effects on histone acetylation profiles, but distinct effects on myeloid progenitor function and neutrophil development. These data suggest that the effects observed in hematopoietic progenitor function are likely to include effects of HDAC inhibitors on non-histone targets. Furthermore, the differences between the effects of TSA, SB and VPA suggest inhibitor specific effects (HDAC preference) or cell specific responses. We are currently investigating the molecular mechanisms underlying these observations.

Description: Signal transducers and activators of transcription (STATs) have been reported to play a critical role in the differentiation of several myeloid cell lines, although the importance of STATs in the differentiation of primary human hematopoietic cells remains to be established. Terminal eosinophil differentiation is induced by interleukin-5 (IL-5), which has also been demonstrated to activate STAT5. We have investigated whether STAT5 plays a critical role during eosinophil differentiation using umbilical cord blood–derived CD34+ cells. In this ex vivo system, STAT5 expression and activation are high early during differentiation, and STAT5 protein expression is down-regulated during the final stages of eosinophil differentiation. Retroviral transductions were performed to ectopically express wild-type and dominant-negative STAT5a (STAT5aΔ750) in CD34+ cells. Transduction of cells with STAT5a resulted in enhanced proliferation compared with cells transduced with empty vector alone. Interestingly, ectopic expression of STAT5a also resulted in accelerated differentiation. In contrast, ectopic expression of STAT5aΔ750 resulted in a block in differentiation, whereas proliferation was also severely inhibited. Similar results were obtained with dominant-negative STAT5b. Forced expression of STAT5a enhanced expression of the STAT5 target genes Bcl-2 andp21WAF/Cip1, suggesting they may be important in STAT5a-mediated eosinophil differentiation. These results demonstrate that STAT5 plays a critical role in eosinophil differentiation of primary human hematopoietic cells.