Hanno Hock

Endogenous oncogenic K-rasG12D stimulates proliferation and widespread neoplastic and developmental defects

Activating mutations in the ras oncogene are not considered sufficient to induce abnormal cellular proliferation in the absence of cooperating oncogenes. We demonstrate that the conditional expression of an endogenous K-ras(G12D) allele in murine embryonic fibroblasts causes enhanced proliferation and partial transformation in the absence of further genetic abnormalities. Interestingly, K-ras(G12D)-expressing fibroblasts demonstrate attenuation and altered regulation of canonical Ras effector signaling pathways. Widespread expression of endogenous K-ras(G12D) is not tolerated during embryonic development, and directed expression in the lung and GI tract induces preneoplastic epithelial hyperplasias. Our results suggest that endogenous oncogenic ras is sufficient to initiate transformation by stimulating proliferation, while further genetic lesions may be necessary for progression to frank malignancy.

Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells

Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion, as a single HSC may reconstitute lethally irradiated hosts. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified, transcription factors restricting their cycling have remained elusive. Here we report that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity.

Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene.

The production of blood cells is sustained throughout the lifetime of an individual by haematopoietic stem cells (HSCs). Specification of HSCs from mesoderm during embryonic development requires the stem cell leukaemia SCL/tal-1 gene product. Forced expression of SCL/tal-1 strongly induces blood formation in embryos, indicating that this gene has a dominant role in commitment to haematopoiesis. In the adult haematopoietic system, expression of SCL/tal-1 is enriched in HSCs and multipotent progenitors, and in erythroid and megakaryocytic lineages, consistent with roles for this factor in adult haematopoiesis. Here we assess by conditional gene targeting whether SCL/tal-1 is required continuously for the identity and function of HSCs. We find that SCL/tal-1 is dispensable for HSC engraftment, self-renewal and differentiation into myeloid and lymphoid lineages; however, the proper differentiation of erythroid and megakaryocytic precursors is dependent on SCL/tal-1. Thus, SCL/tal-1 is essential for the genesis of HSCs, but its continued expression is not essential for HSC functions. These findings contrast with lineage choice mechanisms, in which the identity of haematopoietic lineages requires continuous transcription factor expression.

Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells

Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells1, 2 and have been presumed to retain labels such as the nucleotide analogue 5-bromodeoxyuridine (BrdU). However, recently it has been demonstrated that BrdU-retention is neither sensitive nor specific for HSCs3. Here we show that transient, transgenic expression of a Histone2B (H2B)-Green Fluorescent Protein (GFP) fusion protein in mice allows superior labeling of HSCs and permits improved analysis of their turnover in combination with other markers. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a highly stringent marker combination (L−K+S+CD48−CD150+)4, revealed unexpected heterogeneity in their proliferation rates and suggests that ~ 20% of HSCs turn over at an extremely low rate (≤ 0.8–1.8% per day). Prospective isolation and transplantation of L−K+S+CD48−CD150+ HSCs with different H2B-GFP levels revealed that higher H2B-GFP label retention correlates with superior long-term repopulation potential.Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells and have been presumed to retain labels such as the thymidine analog 5-bromodeoxyuridine (BrdU). However, BrdU retention is neither a sensitive nor specific marker for HSCs. Here we show that transient, transgenic expression of a histone 2B (H2B)–green fluorescent protein (GFP) fusion protein in mice has several advantages for label-retention studies over BrdU, including rapid induction of H2B-GFP in virtually all HSCs, higher labeling intensity and the ability to prospectively study label-retaining cells, which together permit a more precise analysis of division history. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a stringent marker combination (L−K+S+CD48−CD150+), revealed unexpected heterogeneity in their proliferation rates and showed that ∼20% of HSCs divide at an extremely low rate (≤0.8–1.8% per day).

Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4, Sox2, Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however, direct evidence for this notion is lacking. Here, we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do, yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover, we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy.

Intrinsic requirement for zinc finger transcription factor Gfi-1 in neutrophil differentiation.

We report essential roles of zinc finger transcription factor Gfi-1 in myeloid development. Gene-targeted Gfi-1(-/-) mice lack normal neutrophils and are highly susceptible to abscess formation by gram-positive bacteria. Arrested, morphologically atypical, Gr1(+)Mac1(+) myeloid cells expand with age in the bone marrow. RNAs encoding primary but not secondary or tertiary neutrophil (granulocyte) granule proteins are expressed. The atypical Gr1(+)Mac1(+) cell population shares characteristics of both the neutrophil and macrophage lineages and exhibits phagocytosis and respiratory burst activity. Reexpression of Gfi-1 in sorted Gfi-1(-/-) progenitors ex vivo rescues neutrophil differentiation in response to G-CSF. Thus, Gfi-1 not only promotes differentiation of neutrophils but also antagonizes traits of the alternate monocyte/macrophage program.

The Mst1 and Mst2 kinases control activation of rho family GTPases and thymic egress of mature thymocytes.

In mice lacking both Mst1 and Mst2 in the lymphoid compartment, thymocyte development is normal, but single-positive thymocytes exhibit excessive apoptosis and greatly diminished thymic egress, accompanied by loss of chemokine activation of RhoA and Rac1.

A complex Polycomb issue: the two faces of EZH2 in cancer

In the April 1, 2012, issue of Genes & Development, Simon and colleagues (pp. 651-656) demonstrated that the disruption of Ezh2 in mice is sufficient to cause T-acute lymphoblastic leukemia (T-ALL). Moreover, in concert with concurrent studies, the authors revealed that similar mechanisms are involved in human T-ALL. These data contrast with previous findings showing that increased EZH2 activity promotes cancer.

Zinc-finger transcription factor Gfi-1 : versatile regulator of lymphocytes, neutrophils and hematopoietic stem cells

Purpose of reviewGfi-1, originally identified as a transcriptional repressor in lymphoid cells, is now recognized to have essential, independent functions in neutrophil maturation and hematopoietic stem cell biology. Here we review recent studies pertaining to Gfi-1 and its cell context specific functions in hematopoietic development. Recent findingsProgress in mapping the precise timing of requirements for myeloid transcription factors during hematopoietic development reveals a more refined picture of their sequence of action. We contrast Gfi-1s role in neutrophil development with PU.1, C/EBPα, and C/EBPϵ. Gfi-1 has been found to be a major regulator of adult hematopoietic stem cells. It is required for restricting the proliferation of hematopoietic stem cells and maintaining their functional integrity. We discuss its role and compare its function with that of other regulators recently implicated in the biology of hematopoietic stem cells. SummaryConsiderable progress has been made in understanding Gfi-1s context-sensitive roles at defined stages of hematopoietic differentiation.

TEL-AML1 Corrupts Hematopoietic Stem Cells to Persist in the Bone Marrow and Initiate Leukemia

The initial steps in the pathogenesis of acute leukemia remain incompletely understood. The TEL-AML1 gene fusion, the hallmark translocation in Childhood Acute Lymphoblastic Leukemia and the first hit, occurs years before the clinical disease, most often in utero. We have generated mice in which TEL-AML1 expression is driven from the endogenous promoter and can be targeted to specific populations. TEL-AML1 renders mice prone to malignancy after chemical mutagenesis when expressed in hematopoietic stem cells (HSCs), but not in early lymphoid progenitors. We reveal that TEL-AML1 markedly increases the number of HSCs and predominantly maintains them in the quiescent (G(0)) stage of the cell cycle. TEL-AML1(+) HSCs retain self-renewal properties and contribute to hematopoiesis, but fail to out-compete normal HSCs. Our work shows that stem cells are susceptible to subversion by weak oncogenes that can subtly alter their molecular program to provide a latent reservoir for the accumulation of further mutations.