Miranda Buitenhuis

Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia

The cellular targets of primary mutations and malignant transformation remain elusive in most cancers. Here, we show that clinically and genetically different subtypes of acute lymphoblastic leukemia (ALL) originate and transform at distinct stages of hematopoietic development. Primary ETV6-RUNX1 (also known as TEL-AML1) fusions and subsequent leukemic transformations were targeted to committed B-cell progenitors. Major breakpoint BCR-ABL1 fusions (encoding P210 BCR-ABL1) originated in hematopoietic stem cells (HSCs), whereas minor BCR-ABL1 fusions (encoding P190 BCR-ABL1) had a B-cell progenitor origin, suggesting that P190 and P210 BCR-ABL1 ALLs represent largely distinct tumor biological and clinical entities. The transformed leukemia-initiating stem cells in both P190 and P210 BCR-ABL1 ALLs had, as in ETV6-RUNX1 ALLs, a committed B progenitor phenotype. In all patients, normal and leukemic repopulating stem cells could successfully be separated prospectively, and notably, the size of the normal HSC compartment in ETV6-RUNX1 and P190 BCR-ABL1 ALLs was found to be unaffected by the expansive leukemic stem cell population.

The PI3K/PKB signaling module as key regulator of hematopoiesis: Implications for therapeutic strategies in leukemia

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.

Molecular Mechanisms Underlying FIP1L1-PDGFRA–Mediated Myeloproliferation

An interstitial deletion on chromosome 4q12 resulting in the formation of the FIP1L1-PDGFRA fusion protein is involved in the pathogenesis of imatinib-sensitive chronic eosinophilic leukemia. The molecular mechanisms underlying the development of disease are largely undefined. Human CD34(+) hematopoietic progenitor cells were used to investigate the role of FIP1L1-PDGFRA in modulating lineage development. FIP1L1-PDGFRA induced both proliferation and differentiation of eosinophils, neutrophils, and erythrocytes in the absence of cytokines, which could be inhibited by imatinib. Whereas expression of FIP1L1-PDGFRA in hematopoietic stem cells and common myeloid progenitors induced the formation of multiple myeloid lineages, expression in granulocyte-macrophage progenitors induced only the development of eosinophils, neutrophils, and myeloblasts. Deletion of amino acids 30 to 233 in the FIP1L1 gene [FIP1L1(1-29)-PDGFRA] gave rise to an intermediate phenotype, exhibiting a dramatic reduction in the number of erythrocytes. FIP1L1-PDGFRA and FIP1L1(1-29)-PDGFRA both induced the activation of p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) in myeloid progenitors, whereas signal transducers and activators of transcription 5 (STAT5) and protein kinase B/c-akt were only activated by FIP1L1-PDGFRA. Dominant-negative STAT5 partially inhibited FIP1L1-PDGFRA-induced colony formation, whereas combined inhibition of phosphatidylinositol-3-kinase and ERK1/2 significantly reversed FIP1L1-PDGFRA-induced colony formation. Taken together, these results suggest that expression of FIP1L1-PDFGRA in human hematopoietic progenitors induce a myeloproliferative phenotype via activation of multiple signaling molecules including phosphatidylinositol-3-kinase, ERK1/2, and STAT5.

The role of the PI3K-PKB signaling module in regulation of hematopoiesis

Bone marrow homeostasis is controlled by a series of complex cell fate decisions that determine whether hematopoietic stem and progenitor cells divide, differentiate or die. Exquisite control of the stem cell niche is crucial for the correct maintenance of blood cell production, allowing inductive external signals to modulate stem cell self-renewal or increased production of specific blood cell lineages. Since the first bone marrow transplantation more than 40 years ago, research has been focused on understanding the molecular processes regulating hematopoietic stem cell function. While our appreciation of the transcriptional regulators of hematopoiesis has developed considerably, the role of specific intracellular signal transduction modules remains surprisingly undefined. One such module includes phosphatidylinostiol 3-kinase (PI3K) and protein kinase B (PKB/c-akt). The identification and cloning of PI3K and PKB in the early 1990s has resulted in the publication of a large body of literature providing evidence for a crucial role of these molecules in the regulation of proliferation, apoptosis and differentiation. This review will focus on recent studies that have extended our understanding of how PI3K-PKB can regulate the complex process of hematopoiesis.

Lineage-specific activation of STAT3 by interferon-gamma in human neutrophils.

Binding of interferon‐γ (IFN‐γ) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1α. Selective activation of STAT1α at the IFN‐γ receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN‐γ receptor α‐chain and the SH2 domain of STAT1α. We demonstrate that, in addition to STAT1α, STAT3 is also activated by IFN‐γ in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL‐60 cells, although the STAT3 protein was expressed in these cells. The cell type‐specific activation of STAT3 by IFN‐γ was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell‐specific manner, which might result in cell‐specific gene transcription. J. Leukoc. Biol. 65: 391–396; 1999.

Human CD34-Derived Myeloid Dendritic Cell Development Requires Intact Phosphatidylinositol 3-Kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling

Dendritic cells (DCs) are composed of different subsets that exhibit distinct functionality in the induction and regulation of immune responses. The myeloid DC subsets, including interstitial DCs and Langerhans cells (LCs), develop from CD34+ hematopoietic progenitors via direct DC precursors or monocytes. The molecular mechanisms regulating DC development are still largely unknown and mostly studied in mice. Phosphatidylinositol 3-kinase (PI3K) regulates multiple processes in myeloid cells. This study investigated the role of PI3K signaling in the development of human CD34-derived myeloid DCs. Pharmacologic inhibition of PI3K or one of its downstream targets mTOR reduced interstitial DC and LC numbers in vitro. Increased activity of this signaling module by introduction of constitutively active protein kinase B (PKB/c-Akt) increased the yields of human DC precursors in vitro as well as in transplanted β2-microglobulin−/− NOD/SCID mice in vivo. Signaling inhibition during differentiation did not affect the acquisition of a DC phenotype, whereas proliferation and survival strongly depended on intact PI3K–PKB–mTOR signaling. Interestingly, however, this pathway became redundant for survival regulation upon terminal differentiation, which was associated with an altered expression of apoptosis regulating genes. Although dispensable for costimulatory molecule expression, the PI3K–PKB–mTOR signaling module was required for other important processes associated with DC function, including Ag uptake, LPS-induced cytokine secretion, CCR7 expression, and T cell stimulation. Thus, PI3K–PKB–mTOR signaling plays a crucial role in the development of functional CD34-derived myeloid DCs. These findings could be used as a strategy to manipulate DC subset distribution and function to regulate immunity.

Eosinophil differentiation in the bone marrow is inhibited by T cell-derived IFN-γ

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.

Histone deacetylase inhibition modulates cell fate decisions during myeloid differentiation

Background The clinical use of chromatin-modulating drugs, such as histone deacetylase inhibitors, for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last few years. Nonetheless, little is currently known concerning their effects on myelopoiesis. Design and Methods We utilized an ex vivo differentiation system in which umbilical cord blood-derived CD34+ cells were treated with trichostatin A, sodium butyrate and valproic acid to evaluate the effect of histone deacetylase inhibitor treatment on myeloid lineage development, colony-forming potential, proliferation, and terminal neutrophil differentiation. Results Trichostatin A treatment modestly reduced progenitor proliferation, while sodium butyrate and valproic acid resulted in concentration-dependent effects on proliferation and apoptosis. Addition of valproic acid uniquely stimulated CD34+ proliferation. Sodium butyrate treatment inhibited terminal neutrophil differentiation both quantitatively and qualitatively. Addition of 100 μM valproic acid resulted in increased numbers of mature neutrophils with a block in differentiation at increasing concentrations. Sodium butyrate and valproic acid treatment resulted in increased acetylation of histones 3 and 4 while trichostatin A, sodium butyrate and valproic acid had differential effects on the acetylation of non-histone proteins. Conclusions Individual histone deacetylase inihibitors had specific effects on cell fate decisions during myeloid development. These data provide novel insights into the effects of histone deacetylase inhibitors on the regulation of normal hematopoiesis, which is of importance when considering utilizing these compounds for the treatment of myeloid malignancies and bone marrow failure syndromes.

PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function

Plasmacytoid dendritic cells (pDCs) are considered potential tools or targets for immunotherapy. However, current knowledge concerning methodologies to manipulate their development or function remains limited. Here, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mammalian target of rapamycin (mTOR) axis in human pDC development, survival, and function. In vitro pDC generation from human cord blood-derived CD34(+) hematopoietic progenitors was reduced by pharmacologic inhibition of PI3K, PKB, or mTOR activity, and peripheral blood pDCs required PI3K-PKB-mTOR signaling to survive. Accordingly, activity of this pathway in circulating pDCs correlated with their abundance in peripheral blood. Importantly, introduction of constitutively active PKB or pharmacologic inhibition of negative regulator phosphatase and tensin homolog (PTEN) resulted in increased pDC numbers in vitro and in vivo. Furthermore, MHC class II and costimulatory molecule expression, and production of IFN-α and TNF-α, were augmented, which could be explained by enhanced IRF7 and NF-κB activation. Finally, the numerically and functionally impaired pDCs of chronic hepatitis B patients demonstrated reduced PI3K-PKB-mTOR activity. In conclusion, intact PI3K-PKB-mTOR signaling regulates development, survival, and function of human pDCs, and pDC development and functionality can be promoted by PI3K-PKB hyperactivation. Manipulation of this pathway or its downstream targets could be used to improve the generation and function of pDCs to augment immunity.

Protein kinase B (PKB/c-akt) regulates homing of hematopoietic progenitors through modulation of their adhesive and migratory properties

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.