The regulation of the fine balance between physiological differentiation and malignant transformation is one of the most intriguing issues in biology. The development of the hematopoietic system, which is controlled by a plethora of cell intrinsic and extrinsic signals, is an example of this. Slight alterations of signaling strength caused by one or several acquired mutations lead to transformation and the induction of blood tumors, which in turn lead to the development of leukemia or lymphoma.
Initiation of adult hematopoiesis takes place in the bone marrow, where hematopoietic stem cells (HSCs) reside within specialized microenvironmental "niches." HSCs differentiate through intermediate progenitor stages and commit to the myeloid or lymphoid lineages. Most blood sublineages arise in the bone marrow, with the exception of the T cell lineage, which requires the thymus for optimal maturation. The past decade's explosion in the understanding of hematopoietic lineage commitment has led to the identification of an extended complex of transcription factors that orchestrate cell fate decisions. One of the better characterized paradigms was Notch signaling, which proved to be the essential factor leading to the commitment of hematopoietic progenitors to the T cell lineage. Several of these factors, including Notch, subsequently proved to have key oncogenic or tumor-suppressor functions, once more connecting physiological differentiation to malignant transformation.
Our early work has identified Notch and its interaction with T cell receptor (TCR) complexes as essential regulators of T cell progenitor commitment and further differentiation. More specifically, we have studied the essential function of the pre-TCR, a receptor expressed on the surface of immature hematopoietic progenitors in the thymus, and we have identified several important target genes and signaling pathways that control the generation of mature T cells. These genes include regulators of cell cycle entry, cell survival, and TCR rearrangement. Our work has uncovered functional interactions between the pre-TCR and not only Notch but also additional developmentally imprinted pathways, including Wingless and Hedgehog.
Our lab has also shown that the signaling cooperation between the pre-TCR and Notch pathways is clinically important, as it is essential for the induction and establishment of T cell acute lymphoblastic leukemia (T-ALL), a frequent and devastating pediatric blood tumor. We have shown that silencing of the activity of the NFκB pathway or the expression of cyclin D3, both targets of pre-TCR/NFκB signaling, can inhibit T-ALL cell growth and leads to the suppression of disease progression in animal models of T-ALL. Based on these findings, we are developing therapeutic protocols that would target the activity of D-type cyclins and that could be introduced in T-ALL patient clinical trials.
The Notch signaling pathway, a conserved regulator of developmental decisions from flies to humans, took center stage in the study of T cell leukemia when it was shown that the majority of T-ALL patients carry activating mutations on the NOTCH1 gene. Our lab has introduced these mutations in mice and generated faithful disease models that can be used for preclinical drug screening. Using these novel in vivo tools, we recently identified the interaction between the chemokine receptor CCR7 and its ligands CCL19 and CCL21 as an essential "enter" signal for the leukemic cells that cross the brain-blood barrier and infiltrate the central nervous system. These studies underline the importance of cancer modeling and propose new molecularly targeted therapy protocols for the treatment of T cell leukemia.
We have also attempted to identify the molecular mechanism behind mutation-induced pathway activation. More specifically we have demonstrated that one of the mutation hotspots on the C-terminal domain of Notch1 (PEST domain) is the docking site for the E3 ubiquitin ligase Fbw7. Fbw7 has the ability to bind, ubiquitinate, and degrade nuclear Notch1. Furthermore, we have identified two ways by which oncogenic Notch1 can "escape" from Fbw7-mediated degradation: either by mutating and truncating its PEST domain or by inactivating mutations of the Fbw7 ligase. These mutations target specific residues of the Fbw7-binding pocket and lead to the stabilization of Notch and other protein substrates, including c-Myc and cyclin E. Identical mutations were also identified in solid tumors, making Fbw7 a key human tumor suppressor.
Deletion of the Fbw7 gene in hematopoietic progenitor cells led to the induction of T cell malignancy, as predicted from our previous studies. However, Fbw7 deletion in HSCs led to severe anemia, caused by the total loss of HSC quiescence and self-renewal. Further studies have demonstrated that the Fbw7 enzyme is functioning as an HSC cell cycle "brake," ensuring quiescence of adult HSCs. By controlling the cell cycle machinery, Fbw7 indirectly regulates a constellation of genes that are responsible for HSC proliferation, adhesion to the niche, and gene transcription. Current studies using novel in vivo protein-expression reporters suggest that Fbw7 controls all these functions by targeting not Notch1 but c-Myc, another well-characterized protein ubiquitin substrate.
These experiments are introducing us to a novel level of control of stem cell differentiation. They demonstrate that the concerted function of the "ubiquitome" (a group that consists of ubiquitin-conjugating enzymes, ligases, and deubiquitinases among others) is essential for stem cell function and transformation. In our current work, we are mapping expression of ubiquitome members during early stages of hematopoiesis and developing novel genomic, genetic, and proteomic tools to study their function in the regulation of life and death of the immune system.
Grants from the National Institutes of Health, the National Cancer Institute, the American Cancer Society, and the Leukemia and Lymphoma Society provided partial support for these projects.
As of May 30, 2012