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  • Research and Clinical Interests

    Our laboratory studies the molecular mechanisms controlling hematopoietic stem cell (HSC) self-renewal, differentiation and transformation. More specifically we focus on the regulation of HSC cell cycle entry and on the effects of Notch signaling in stem cell differentiation. Finally, we study the role of the Notch pathway as an oncogenic trigger in acute lymphoblastic leukemia. Our goal is to design novel therapies that will target and suppress Notch transforming activity in vivo.

  • CURRENT PROJECTS:

    The E3 ligase Fbw7 as a T-ALL tumor suppressor.

    We have recently shown (Thompson et al. J. Exp. Med. 2007) that the T-ALL oncogene Notch1 is controlled by the E3 ligase Fbw7 (SEL-10, Ago). Fbw7 can bind a conserved “degron” sequence on the Notch1 PEST domain, ubiquitylate the protein and target it for proteasomal degradation. We have shown that Notch1 PEST mutations found in T-ALL patients target this Fbw7 degron and generate stable forms of the Notch1 transcription factor. We have also shown that Fbw7 is by itself inactivated by mutations in a significant fraction of T-ALL patients. Fbw7 mutations generate enzymes that are not able to bind and ubiquitylate several well-characterized substrates including Notch1, c-Myc and cyclin E. We are currently addressing the oncogenic potential of Fbw7 inactivation by generating T cell specific Fbw7 knock-out animals. Moreover, to address the importance of Nocth1 stabilization in T-ALL, we are generating mice that carry Notch1 mutants that lack the conserved Fbw7 degron located in the Notch1 PEST domain. Finally, we are using transcriptome-based approaches to study the signaling pathways affected by Fbw7 inactivation and are exploring genetic and pharmacological ways to regulate the ubiquitin machinery in tumor cells.
    (Lab Members Involved: Ben, Silvia, Thomas)

    The Role of Notch1 in the induction and establishment in T cell acute lymphoblastic leukemia (T-ALL).
    Our lab has shown that oncogenic Notch1 activation in T-ALL is able to target and induce the NF-kB signaling pathway (Vilimas et al. Nature Medicine 2007). Indeed, we have shown that Notch1 mutant expression can activate the IKK kinase and induce the expression of several well-characterized NF-kB gene targets. T-ALL lines carrying Notch1 mutations have shown a constitutive activation of the NF-kB pathway. Upon NF-kB silencing the cells enter rapid apoptosis. Consistent with these findings, attenuation of the NF-kB pathway can target disease progression in a mouse model of Notch1-induced T-ALL. Our current efforts have several targets. Initially, we are using genetic means to study the detailed role of the IKK activation in T-ALL. Also, we are using whole-genome siRNA screening approached to identify factors that can influence the Notch1/NF-kB interaction. Moreover, we are interested in the identification of specific gene-targets of the Notch1/NF-kB pathway that can be important for disease progression. We have recently identified an adhesion molecule that is induced by oncogenic Notch1 activation and is essentially for proper leukemic cell migration and tissue infiltration. Such target identification is of unique importance as it will give us the ability to design molecularly- targeted therapies that will suppress both disease induction and maintenance. Finally, we believe that NF-kB activation can be an attractive theurapeutic target. We have initiated, in collaboration with the NYU Cancer Institute, drug screens using mouse T-ALL models as preclinical screening tools.
    (Lab Members Involved: Severine, Silvia, Thomas, Suqing).

    Notch1 signaling and tumor cell migration/ metastasis.

    T-cell acute lymphoblastic leukemia (T-ALL) is associated with lower remission rates and a greater likelihood of relapse, particularly within the central nervous system (CNS) than standard risk ALL. Over 50% of T-ALL patients have activating mutations in the Notch1 protein. We found that Notch1 activates the NF-kB pathway, which in turn causes the up-regulation several migration and adhesion proteins that have been shown to play a key role in lymphocyte homing in extramedullary tissues and CNS. Using BM infection and transplantation of WT or knockout mice we will pin point the infiltration mechanism used by these very aggressive leukemic cells. In addition, this study will allow us to identify new possible targets for the development of therapy against the infiltration process, with particular attention to CNS involvement.  
    (Lab Members Involved: Silvia, Thomas).

    Regulation of stem cell differentiation by the Fbw7 Ubiquitin ligase.

    Control of protein stability by ubiquitin-dependent proteolysis regulates numerous cellular processes, including a cell’s ability to enter and exit the cell cycle.  Hematopoietic stem cells (HSCs) must carefully restrict their cell cycle entry, as their ability to sustain hematopoiesis throughout an organism’s life requires them to exist in a mostly quiescent state.  HSCs adopt and maintain this unique fate through their interactions with a specialized bone marrow microenvironment called the niche.  Several cell cycle regulators have been implicated as effectors of niche-derived signals, including c-Myc, cyclin E, and Notch. Previous work, including ours, has shown that the ubiquitin ligase, Fbw7, targets all of these proteins for destruction by the proteasome, and in doing so can antagonize cell cycle entry and progression.  We tested the role of Fbw7 in HSC biology by generating conditional knock-out animals in which Fbw7 can be inactivated in a tissue-specific manner.  We found that deletion of Fbw7 from hematopoietic cells using the Mx1-Cre system resulted in a rapid and severe defect in the HSC compartment characterized by a loss of quiescence, particularly in long term (LT) HSCs.  These cells suffer a complete loss of long term repopulating potential both in vitro and in vivo.  Whole transcriptome studies uncovered a large subset of genes associated with “stemness” in LT-HSCs that are significantly down-regulated in the absence of Fbw7, suggesting that loss of Fbw7 function results in a transcriptional reprogramming of HSCs coincident with and/or causative of their exit from quiescence.  We are currently working to understand the specific mechanisms by which Fbw7 maintains HSC quiescence, and to identify the relevant Fbw7 target proteins that, directly or indirectly, orchestrate a transcriptional stemness program in HSCs.
    (Lab Members Involved: Ben, Jie, Alan, Suqing).

    The Role of D-Type cyclins in hematopoeisis

    We have previously shown that cyclin D3 is essential for regulation of proliferation in early lymphocytes (Cooper et al Nature Immunology 2006; Sicinska et al Cancer Cell 2003).   In the absence of D3, both B and T cell development are impaired, due to a reduction in proliferation of pre-B and pre-T cells. This D3-mediated proliferation in pre-B cells is induced by the pre-BCR. This induction does not lead to an increase in D3 transcription but rather inhibits cyclin D3 from degradation via the proteasome. In order to investigate the role of cyclin D3 stability in lymphoid development and leukemogenesis, cyclin D3 mutants have been generated. We also have reported that in the absence of D3, cyclin D2, but not D1, is highly upregulated.  Given the requirement for D3 in early lymphocytes, the high expression of D2 suggests that cyclin D3 has unique functions and cyclin D2 and D3 are not functionally redundant in early lymphocytes.  To further define the roles of cyclins D2 and D3, we have generated cyclin knock-in mice.  Finally, we are identifying the downstream targets of cyclin D3 that are critical for proliferation of early lymphocytes. 
    (Lab Members Involved: Katie, Kelly)