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.

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014

MicroRNA 21 is a novel oncogene in Notch1-driven acute lymphoblastic T cell leukemia

Fabian Junker

Notch signaling is a conserved cell-to-cell communication pathway essential in the development and maintenance of various tissues. Upon ligand binding, Notch receptors on the cell surface are proteolytically cleaved, generating the intracellular domain of Notch (NICD) which acts as a transcriptional regulator. Signaling through Notch1 is essential for normal T cell development, and de-regulated Notch1 signaling leads to the development of acute lymphoblastic T cell leukemia (T-ALL). Mouse models of retroviral overexpression of N1ICD in hematopoietic progenitors in bone-marrow (BM) chimeric mice clearly show that Notch1 in T-ALL acts as an oncogene. MicroRNAs (miRNAs) are small RNA molecules that function as post-transcriptional negative regulators of gene expression. They have been described to play either oncogenic or tumor suppressive roles in Notch1-driven T-ALL. However, it is currently unknown whether miRNAs are essential in Notch1-driven leukemogenesis or in T-ALL maintenance. Dicer1 is an essential enzyme for the generation of mature, functional miRNAs. In this study, the global requirement for miRNAs in T-ALL was assessed via conditional ablation of Dicer1 during early N1ICD-mediated leukemogenesis and in later stages of established T-ALL in vivo. Interestingly, both T-ALL development and maintenance in N1ICD overexpressing BM chimeras were dependent on Dicer1. This was found to be due to induction of apoptosis in Dicer1-deficient T-ALL cells. Microarray-based expression analysis demonstrated that potentially oncogenic miRNAs were up-regulated in both mouse and human T-ALL samples compared with normal T cell progenitors. Among the abundantly expressed miRNAs, candidates previously linked to T-ALL were detected as well as novel candidates. Among them, miR-21, which has previously been linked to tumor biology in a variety of solid tumors, was found to be the most differentially regulated in T-ALL and early and late T cell progenitors. It was also found to be abundantly expressed in primary mouse T-ALL specimens as well as human T-ALL cell lines and primary patient T-ALL samples. Using a novel lentivirus-based miRNA activity reporter system, I demonstrated that miR-21 is active in T-ALL cell lines both in vitro and in vivo. In addition, KD of miR-21 led to apoptosis in T-ALL cells with high miR-21 activity, consistent with miR-21 target gene de-repression. Taken together, these findings demonstrate for the first time that miR-21, in the context of Notch1-driven T-ALL, acts in an oncogenic fashion. It does so by facilitating T-ALL cell survival, which is, at least in part, due to repression of programmed cell-death protein 4 (Pdcd4), a miR-21 target gene and potential novel tumor suppressor in T-ALL. In summary, this study shows that in Notch1-driven T-ALL, miRNAs play an essential role by facilitating T-ALL cell survival, and that miR-21 is a novel oncogenic miRNA in this disease.

EPFL2014

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.

EPFL2011

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

EPFL2011

MicroRNA 21 is a novel oncogene in Notch1-driven acute lymphoblastic T cell leukemia

Fabian Junker

EPFL2014

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

EPFL2014