(T-cell Acute Lymphoblastic Leukemia) Notch1 And T-All (T-cell Acute Lymphoblastic Leukemia) Katie Lozada Hello, my name is Katie Lozada and today I will discuss the Notch1 gene and its role in T-cell Acute Lymphoblastic Leukemia.

How Notch was discovered -Discovered in Drosophila 1917 by Thomas Hunt Morgan -Notch pathway is highly conserved between Drosophila and humans The Notch gene was first discovered in 1917 by Thomas Hunt Morgan. This gene was identified in Drosophila that had notches at the ends of their wing blades. After studying these flies, they have found that it is due to a single copy of a defective Notch gene (-/- are inviable). The Notch gene plays an important role in development of the fruit fly. After sequencing, researchers found a homologous gene in humans. The notch pathway is highly conserved between Drosophila and humans. The pathway controls cell fate decisions during development of both humans and flies.

Notch1 is a cell surface receptor -plays a key role in cell fate decisions Today I focus on Notch1’s importance in the pathway. Notch1 is a transmembrane cell surface receptor. Notch1 is a receptor for membrane bound ligands that sit adjacent to the Notch receptor. The Notch1 protein is one of four receptors that are seen in the Notch pathway

Overview of the Notch Pathway Ligand binds to Notch extracellular domain  induces proteolytic cleavage  releases intracellular domain  enters nucleus, activate CSL  alter gene expression! The Notch signaling pathway begins when a ligand binds to the extracellular domain of the receptor protein. This induces a proteolytic cleavage which releases the intracellular domain of the protein by gamma secretase enzyme. The intracellular domain of the Notch protein now enters the nucleus to activate the transcription factor, CSL.

Overview of Notch Pathway In this diagram, you can see that when a ligand that is adjacent to the Notch receptor binds, it releases the intracellular domain and activates the transcription factor to transcribe certain genes. Maillard et al. Fig 1.

Notch Signaling can have diverse effects Stem Cells Bipotent Thymocytes Tcell Notch signaling has different effects depending on the cell. In stem cells, the Notch pathway inhibits differentiation and keeps the cells in undifferentiated state. In Bipotent thymocyte cells, Notch signaling induces a T-cell fate and blocks B-cell fate. Bcell Inhibit Differentiation Promote Tcell fate Radtke et al. Fig 2.

Notch Signaling can have diverse effects Skin Cells Tcells In transit-amplifying cells, like skin, the Notch signal induces terminal differentiation. In T-cell tumorigenesis, Notch acts an oncogene resulting in proliferation. Terminal differentiation ONCOGENE Radtke et al. Fig 2.

Notch1 knockout mice die at E 9.5 with defects in forming somites This precluded analysis of its role in the lymphocyte lineage Early experiments showed that functional Notch1 is essential for development. Researchers found that Notch is required for complete somite segmentation. As we can see in the picture, the head of the mutant embryo develops normally but the embryo is deficient in posterior elongation. We can only see a few somites.   Since we cannot perform simple knockout experiments, researchers had to find a way to selectively induce the mutant Notch phenotype.

Specifically knock out Notch 1 in adult mice After its roles in embryonic development are over Since we cannot perform simple knockout experiments, researchers had to find a way to selectively induce the mutant Notch phenotype.

Interferon Promoter Korf et al. Fig. 4-17. They induced inactivation of Notch1 after completion of development using Cre Recombinase, which is a bacterial enzyme that mediates site-specific recombination. First, the Notch1 coding sequence was flanked by inserting Iox P gene. The Cre gene was put under control of a promoter that is only active when exposed to interferon. Since interferon is a signaling molecule present in the immune response, the embryos were kept in a pathogen free environment so that the Cre Recombinase could not be activated. Once development had completed, the adult mice were injected with interferon to induce transcription of the Cre gene. The active Cre Recombinase caused recombination of the IoxP and deletes the Notch1 gene. Interferon Promoter Korf et al. Fig. 4-17.

Loss of Notch1 in adults leads to reduction in size of thymus The results of this knockout experiment showed severe growth retardation in the thymus, where lymphocytes mature into T-cells. The knockout mice had defective T-cell development (which was found by additional experiments).

In the absence of Notch, T cell development is blocked Wild type cells allow the lymphocyte precursor to develop into T-cell.   In the Notch1 -/- mutant a cell, the precursors instead became B cells. MacDonald et al.Fig.2.

Notch plays multiple roles in T cells Commitment to T-cell Lineage: -inactivated Notch1  no T cells, accumulate B -constitutively active Notch1  no B cells, accumulate T When Bone Marrow cells were retrovirally transduced with inactivated Notch1, there was an early block in T-cell development, resulting in accumulation of B-cells. When Bone marrow cells were retrovirally transduced with constitutively active Notch1, there was no B cell production but an accumulation of T-cells.

Notch plays multiple roles in T cells This figure shows Notch1’s role in intrathymic T-cell differentiation. As you can see, Notch receptors are important in each step of thymocyte maturation. Grabher et al. Fig 3.

Oncogenic Activity of Notch When constitutively active, Notch1 behaves as an oncogene. It results in a block of differentiation, promotion of survival and increased proliferation. Maillard et al. Fig. 1

Oncogenic Activity of Notch1 Oncogenic signal  differentiation block inhibition of cell cycle arrest and apoptosis This was shown by an inhibition of cell cycle arrest and apoptosis.

T-ALL is a cancer of Lymphoblasts -Uncontrolled T-cell Proliferation -TCR breakpoints in 30%-35% of T-ALL cases During lymphoblast development, mutations arrive leading to various lymphoblastic leukemias. T-ALL is a leukemia of the T-cells. In 30-35% of T-ALL cases, chromosomal breakpoints are found.

Translocation causes constitutively active Notch1 Translocation between Notch1 gene and TCRβ locus These breakpoints result in a translocation between Notch1 and TCRbeta locus. The translocation causes the TCRb promoter to enhance transcription of the Notch1 gene and make it constitutively active. Constitutively active

Notch Tumorigenesis -maintains precursor cells in proliferating and undifferentiated state This constitutively active expression maintains T-cell precursor cells in a proliferating and undifferentiated state.

T-cell Acute Lymphoblastic Leukemia Notch activation leads to activation of as yet unknown genes involved in T cell fate It’s constitutive activation leads to  T cell proliferation and malignancy The genes that the activation induces are not yet known. All we do know is that it leads to cancer of the T-cells.   This picture shows the proliferation of undifferentiated cells.

T-cell Acute Lymphoblastic Leukemia -T-ALL accounts for 10-15% of diagnosed Acute Lymphoblastic Leukemias -Mainly diagnosed in children (age 2-5) -Affects boys more than girls -Associated with large thymus This cancer is a subset of Lymphoblastic Leukemia that progresses rapidly and affects immature blood cells. This disease is diagnosed in children between 2 and 5 years old. Patients with T-ALL have a swollen thymus which can result in difficulty breathing.

Treatment for T-ALL -High survival rate among treated patients -Systemic disease requires multi-drug chemotherapy -High survival rate among treated patients If untreated, T-ALL can progress quickly and result in death. Luckily, there are treatments available to control this disease. Chemotherapy is the main treatment options for those diagnosed with T-cell Acute Lymphoblastic Leukemia.