Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21-mediated tumor suppressor Pdcd4 repression

The modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage- tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T- ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4.

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

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

Fabian Junker

EPFL2014

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

Caroline Poisson

EPFL2011