Hugh J. M. Brady

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.

Mcl-1 Interacts with Truncated Bid and Inhibits Its Induction of Cytochrome c Release and Its Role in Receptor-mediated Apoptosis

Engagement of death receptors such as tumor necrosis factor-R1 and Fas brings about the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria to activate Bax/Bak, resulting in the release of cytochrome c. The mechanism underlying the activation, however, is not fully understood. Here, we have identified the anti-apoptotic Bcl-2 family member Mcl-1 as a potent tBid-binding partner. Site-directed mutagenesis reveals that the Bcl-2 homology (BH)3 domain of tBid is essential for binding to Mcl-1, whereas all three BH domains (BH1, BH2, and BH3) of Mcl-1 are required for interaction with tBid. In vitro studies using isolated mitochondria and recombinant proteins demonstrate that Mcl-1 strongly inhibits tBid-induced cytochrome c release. In addition to its ability to interact directly with Bax and Bak, tBid also binds Mcl-1 and displaces Bak from the Mcl-1-Bak complex. Importantly, overexpression of Mcl-1 confers resistance to the induction of apoptosis by both TRAIL and tumor necrosis factor-α in HeLa cells, whereas targeting Mcl-1 by RNA interference sensitizes HeLa cells to TRAIL-induced apoptosis. Therefore, our study demonstrates a novel regulation of tBid by Mcl-1 through protein-protein interaction in apoptotic signaling from death receptors to mitochondria.

The transcription factor E4bp4/Nfil3 controls commitment to the NK lineage and directly regulates Eomes and Id2 expression

E4bp4 is required for commitment to the NK lineage and promotes NK development by directly regulating the expression of Eomes and Id2.

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.

Differential Requirement for Nfil3 during NK Cell Development

NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow–derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL+ Eomes− NK cells develop independently of Nfil3. Loss of Nfil3 during the development of bone marrow–derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3−/− progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3.

Characterisation of Mcl-1 cleavage during apoptosis of haematopoietic cells

Mcl‐1 is essential for normal haematopoiesis, being required for lymphocyte development and maintenance. Its role in haematopoietic differentiation and development is associated with its function as an anti‐apoptotic member of the Bcl‐2 family of proteins although the underlining mechanism is poorly understood. We have characterized caspase cleavage of the Mcl‐1 protein during apoptosis. Caspase cleavage resulted in the removal of the PEST regions from the protein and generation of a fragment containing the BH‐1, ‐2 and ‐3 homology domains. Removal of the PEST regions did not appear to alter Mcl‐1 stability, suggesting that these regions are not responsible for Mcl‐1s short half‐life. In addition, unlike cleavage of Bcl‐2 and Bcl‐XL, which resulted in pro‐apoptotic fragments, cleaved forms of Mcl‐1 were unable to induce apoptosis. This novel regulation of Mcl‐1 may have important implications not only for its role in apoptosis but also for the essential role it plays in the differentiation and development of haematopoietic cells.

Adipose Natural Killer Cells Regulate Adipose Tissue Macrophages to Promote Insulin Resistance in Obesity

Obesity-induced inflammation mediated by immune cells in adipose tissue appears to participate in the pathogenesis of insulin resistance. We show that natural killer (NK) cells in adipose tissue play an important role. High-fat diet (HFD) increases NK cell numbers and the production of proinflammatory cytokines, notably TNFα, in epididymal, but not subcutaneous, fat depots. When NK cells were depleted either with neutralizing antibodies or genetic ablation in E4bp4(+/-) mice, obesity-induced insulin resistance improved in parallel with decreases in both adipose tissue macrophage (ATM) numbers, and ATM and adipose tissue inflammation. Conversely, expansion of NK cells following IL-15 administration or reconstitution of NK cells into E4bp4(-/-) mice increased both ATM numbers and adipose tissue inflammation and exacerbated HFD-induced insulin resistance. These results indicate that adipose NK cells control ATMs as an upstream regulator potentially by producing proinflammatory mediators, including TNFα, and thereby contribute to the development of obesity-induced insulin resistance.

E4BP4: an unexpected player in the immune response.

Until recently, the basic leucine zipper transcription factor E4BP4 (also known as NFIL3) was of little interest to immunologists, being best known for its role in regulating circadian rhythm in chick pineal gland. However, characterisation of E4bp4(-/-) mice, independently generated in four different laboratories, has revealed roles for E4BP4 in diverse haematopoietic lineages. E4BP4 is essential for the development of NK cells and CD8α(+) conventional dendritic cells, and is also involved in macrophage activation, polarisation of CD4(+) T cell responses and B cell class switching to IgE. Here, we discuss the role of E4BP4 as a regulator of the immune response and highlight future questions for the field.

CD8α+ DCs can be induced in the absence of transcription factors Id2, Nfil3, and Batf3

Antiviral immunity and cross-presentation is mediated constitutively through CD8α+ and CD103+ DCs. Development of these DC subsets is thought to require the transcription factors Irf8, Id2, Nfil3, and Batf3, although how this network is regulated is poorly defined. We addressed the nature of the differentiation blocks observed in the absence of these factors and found that although all 4 factors are required for CD103+ DC development, only Irf8 is essential for CD8α+ DCs. CD8α+ DCs emerged in the absence of Id2, Nfil3 and Batf3 in short-term bone marrow reconstitution. These “induced” CD8α+ DCs exhibit several hallmarks of classic CD8α+ DCs including the expression of CD24, Tlr3, Xcr1, Clec9A, and the capacity to cross-present soluble, cell-associated antigens and viral antigens even in the absence of Batf3. Collectively, these results uncover a previously undescribed pathway by which CD8α+ DCs emerge independent of Id2, Nfil3, and Batf3, but dependent on Irf8.

Ataxia Telangiectasia Mutated and p21CIP1 Modulate Cell Survival of Drug-Induced Senescent Tumor Cells: Implications for Chemotherapy

Purpose: Premature or stress-induced senescence is a major cellular response to chemotherapy in solid tumors and contributes to successful treatment. However, senescent tumor cells are resistant to apoptosis and may also reenter the cell cycle. We set out to find a means to specifically induce senescent tumor cells to undergo cell death and not to reenter the cell cycle that may have general application in cancer therapy. Experimental Design: We investigated the mechanisms regulating cell survival in drug-induced senescent tumor cells. Using immunofluorescence and flow cytometry–based techniques, we established the status of the ataxia telangiectasia mutated (ATM) signaling pathway in these cells. We assayed the requirement of ATM signaling and p21CIP1 expression for survival in premature senescent tumor cells using pharmacologic inhibitors and antisense oligonucleotides. Results: The ATM/ATR (ATM- and Rad3-related) signaling pathway was found to be constitutively active in drug-induced senescent tumor cells. We found that blocking ATM/ATR signaling with pharmacologic inhibitors, including the novel ATM inhibitors KU55933 and CGK733, induced senescent breast, lung, and colon carcinoma cells to undergo cell death. We show that the mechanism of action of this effect is directly via p21CIP1, which acts downstream of ATM. This is in contrast to the effects of ATM inhibitors on normal, untransformed senescent cells. Conclusions: Blocking ATM and/or p21CIP1 following initial treatment with a low dose of senescence-inducing chemotherapy is a potentially less toxic and highly specific treatment for carcinomas.