Hes1 as a Mediator of Notch Signalling in T-cell Development and Notch1- Induced T-ALL

The evolutionarily conserved Notch signalling pathway controls a broad spectrum of cell fate decisions in organisms as diverse as fruit flies and mice. Whithin the murine haematopoietic system, Notch1 is indispensable for T cell development as conditional inactivation of Notch1 in bone marrow (BM) precursors results in a complete block in T-cell development and ectopic B-cell development in the thymus. Conversely, constitutive activation of Notch1 signalling plays a causative role in the development of acute T-cell lymphoblastic leukaemia (T-ALL). Numerous mutations within the Notch1 gene, which result in aberrent activation of Notch1 signalling, have been identified in > 50% of human T-ALL patients. Therefore, gain-of-function studies in murine models of T-ALL have been employed by many laboratories to elucidate the molecular mechanisms acting either cooperatively or downstream of oncogenic Notch1 signalling in development and progression of T-ALL. The best-characterized Notch1 target gene to date is a member of the bHLH family of transcriptional repressors, Hes1. This study focuses on the role of Hes1 as a mediator of Notch1 signalling in adult murine haematopoietic development, as well as the requirement for Hes1 in T-ALL development and maintenance. Conditional inactivation of Hes1 in adult murine BM cells results in severe thymic hypoplasia, while myeloid and B cell development remain unperturbed. Upon transplantation in congenic recipients, Hes1-/- progenitors give rise to only a small number of mature T cells. Hes1-inactivation does not however cause ectopic B-cell development in the thymus, and therefore does not phenocopy the loss of Notch1. Thus, while Notch1 signalling is indispensable for T versus B-cell fate specification, this function is not mediated exclusively via Hes1. Strikingly, in a competitive BM chimera setting, Hes1-/- progenitors completely fail to generate T cells. The mechanism by which Hes1 inactivation results in depleted T cell numbers remains to be elucidated, although we have shown that loss of Hes1 does not result in enhanced cell death. In addition, preliminary data are indicative of cell-cycle perturbation in Hes1-deficient immature T cells. Finally, we have demonstrated that upon IT transplantation, Hes1-deficient progenitors successfully give rise to T-cells at all stages of development. This data strongly indicates that Hes1 deficiency does not result in an intrinsic developmental defect but rather in the inability of T cell progenitors to efficiently home to the thymus from the BM. Upregulation of Hes1 expression has been observed in both human T-ALL cell lines as well as murine primary Notch1-induced T-ALL samples. However, the function of Hes1 as an effector of oncogenic Notch1 signalling in modulating disease onset or progression remained to be investigated. The requirement for Hes1 as a mediator of Notch1-induced T-ALL was addressed by inducing simultaneous deletion of Hes1 and expression of a dominant active form of Notch1 (NICD) in a murine model of T-ALL. Our data demonstrate that Hes1 is essential for the development of NICD-induced T-ALL. Loss of Hes1 prevents ectopic development of DP (CD4+CD8+) leukemic T cells in the BM and the subsequent accumulation of DP T cells in peripheral tissues, a hallmark of T-ALL, and consequently results in 10-fold increased life expectancy of experimental animals. We have further determined that Hes1 is required for the maintenance of T-ALL by inactivating Hes1 in a retrovirally pre-induced T-ALL model. Deletion of Hes1 in leukemic DP cells results in rapid cell death and enhanced survival. These data demonstrate that Hes1 is required as a mediator of Notch1 signalling in normal T cell development, and is indispensable to mediate the oncogenic effect of constitutive Notch1 activation in the induction of T-ALL.