Owen Williams

Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal.

The Mixed Lineage Leukemia (Mll) gene is a homolog of Drosophila Trithorax commonly rearranged in infant leukemia. Comprehensive analysis of the role of Mll in hematopoiesis in fetal and adult knockout mice has been prevented by the lethality of Mll(-/-) mice. We have established a conditional deletion model that allows us to study adult hematopoiesis in the absence of Mll. In this study, Mll(-/-) embryos survive to E16.5 and have reduced numbers of HSCs. The quiescent fraction of these HSCs is greatly reduced, and they are unable to compete with wild-type cells in transplantation assays. Mice with Mll expression conditionally deleted in the hematopoietic system have grossly normal hematopoiesis in bone marrow, thymus, and spleen. However, transplanted Mll-deficient bone marrow cells are highly compromised in their ability to competitively reconstitute irradiated recipients. These results suggest a critical role for Mll in regulating stem cell self-renewal.

Anti-γδ T cell antibody blocks the induction and maintenance of oral tolerance to ovalbumin in mice

Abstract The oral administration of antigens is one of the means of inducing tolerance in adult mammals. In this report, the role of γδ T cells in the induction and maintenance of orally-induced tolerance to ovalbumin was investigated. The injection of a monoclonal anti-γδ T cell monoclonal antibody blocked the induction of oral tolerance, because the secondary immune responses to ovalbumin in these animals were comparable to the corresponding responses in ovalbumin-immunized control mice. Furthermore, depletion of γδ T cells either in vivo or in vitro abolished already established oral-tolerance. The fact that the state of tolerance could be adoptively transferred to naive recipients by CD3+αβ−γδ+ spleen cells from tolerant mice. These results suggest that systemic oral tolerance is induced and actively maintained by mechanisms involving γδ T cells.

ERG Is a Megakaryocytic Oncogene

Ets-related gene (ERG) is a member of the ETS transcription factor gene family located on Hsa21. ERG is known to have a crucial role in establishing definitive hematopoiesis and is required for normal megakaryopoiesis. Truncated forms of ERG are associated with multiple cancers such as Ewings sarcoma, prostate cancer, and leukemia as part of oncogenic fusion translocations. Increased expression of ERG is highly indicative of poor prognosis in acute myeloid leukemia and ERG is expressed in acute megakaryoblastic leukemia (AMKL); however, it is unclear if expression of ERG per se has a leukemogenic activity. We show that ectopic expression of ERG in fetal hematopoietic progenitors promotes megakaryopoiesis and that ERG alone acts as a potent oncogene in vivo leading to rapid onset of leukemia in mice. We observe that the endogenous ERG is required for the proliferation and maintenance of AMKL cell lines. ERG also strongly cooperates with the GATA1s mutated protein, found in Down syndrome AMKL, to immortalize megakaryocyte progenitors, suggesting that the additional copy of ERG in trisomy 21 may have a role in Down syndrome AMKL. These data suggest that ERG is a hematopoietic oncogene that may play a direct role in myeloid leukemia pathogenesis.

Hsa-mir-125b-2 is highly expressed in childhood ETV6/RUNX1 (TEL/AML1) leukemias and confers survival advantage to growth inhibitory signals independent of p53

MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT–PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: sensitivity of human thymocytes.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

In focus: MLL-rearranged leukemia

The molecular mechanisms underlying oncogenesis in leukemias associated with rearrangement of the Mixed Lineage Leukemia (MLL) gene have received a considerable amount of attention over the last two decades. In this review we will focus on recent studies, published over the past year, that reveal new insights into the multi-protein complexes formed by MLL and MLL fusion proteins, the role of epigenetic deregulation in MLL fusion function, downstream transcriptional target genes, the importance of the leukemia cell of origin, the role played by microRNAs, cooperating mutations and the implications that recent research has for the therapy of MLL-rearranged leukemia.

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes

Glucocorticoids have significant immunoregulatory actions on thymocytes and T cells and act by binding and activating cytosolic glucocorticoid receptors, which translocate to the nucleus and control gene expression through binding to specific response elements in target genes. Glucocorticoids promote cell death by activating an apoptotic program that requires transcriptional regulation. We set out to identify genes that are crucial to the process of glucocorticoid-mediated thymocyte apoptosis. Freshly isolated murine primary thymocytes were treated with dexamethasone, mRNA isolated and used to screen DNA microarrays. A set of candidate genes with upregulated expression was identified and selected members assayed in reconstituted fetal thymic organ culture (FTOC). Fetal liver-derived hematopoietic progenitor cells (HPCs) were infected with retroviruses expressing individual genes then used to repopulate depleted fetal thymic lobes. Reconstituted FTOCs expressing the gene Tnfaip8 were treated with dexamethasone and shown to be greatly sensitized to dexamethasone. Retrovirus-mediated RNA interference was applied to knock down Tnfaip8 expression in HPCs and these were used to reconstitute FTOCs. We observed that downregulating the expression of Tnfaip8 alone was sufficient to effectively protect thymocytes against glucocorticoid-induced apoptosis. We propose that Tnfaip8 is crucial in regulating glucocorticoid-mediated apoptosis of thymocytes.

Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia.

Decreased autophagy contributes to malignancies; however, it is unclear how autophagy has an impact on tumor growth. Acute myeloid leukemia (AML) is an ideal model to address this as (i) patient samples are easily accessible, (ii) the hematopoietic stem and progenitor cells (HSPC) where transformation occurs is well characterized and (iii) loss of the key autophagy gene Atg7 in HSPCs leads to a lethal pre-leukemic phenotype in mice. Here we demonstrate that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared with more committed/mature hematopoietic cells, healthy human and mouse HSPCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Taken together, with our previous findings these data are compatible with autophagy-limiting leukemic transformation. In line with this, autophagy gene losses are found within chromosomal regions that are commonly deleted in human AML. Moreover, human AML blasts showed reduced expression of autophagy genes and displayed decreased autophagic flux with accumulation of unhealthy mitochondria, indicating that deficient autophagy may be beneficial to human AML. Crucially, heterozygous loss of autophagy in an MLL–ENL model of AML led to increased proliferation in vitro, a glycolytic shift and more aggressive leukemias in vivo. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy, providing a general advantage for tumor growth.

CBFβ is critical for AML1-ETO and TEL-AML1 activity

AML1-ETO and TEL-AML1 are chimeric proteins resulting from the t(8;21)(q22;q22) in acute myeloid leukemia, and the t(12;21)(p13;q22) in pre-B-cell leukemia, respectively. The Runt domain of AML1 in both proteins mediates DNA binding and heterodimerization with the core binding factor beta (CBFbeta) subunit. To determine whether CBFbeta is required for AML1-ETO and TEL-AML1 activity, we introduced amino acid substitutions into the Runt domain that disrupt heterodimerization with CBFbeta but not DNA binding. We show that CBFbeta contributes to AML1-ETOs inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and is indispensable for its cooperativity with the activated receptor tyrosine kinase TEL-PDGFbetaR in generating acute myeloid leukemia in mice. Similarly, CBFbeta is essential for TEL-AML1s ability to promote self-renewal of B cell precursors in vitro. These studies validate the Runt domain/CBFbeta interaction as a therapeutic target in core binding factor leukemias.

The agonist-antagonist balance in positive selection

Peptide antigens expressed in the thymus, in combination with self major histocompatibility complex molecules play a crucial role in thymocyte selection and shaping of the mature T-cell repertoire. Here, it is proposed that a single thymocyte may be exposed to numerous different peptide ligands as it matures, such that its fate is determined by the sum of signals produced by these interactions.