Role of the Notch signalling pathway in acute myeloid leukaemia

We are trying to identify the role that a gene called Notch plays in acute leukaemia. We already know that over half of children with T-cell acute lymphoblastic leukaemia (T-ALL) have mutations in Notch-1, but their role in the causation of the disease is not completely understood.

The Notch pathway plays several roles in normal blood cell production, being particularly important in the production of T-cells and blood stem cells.

Leukaemic cells are unable to develop into normal blood cells. Our preliminary studies suggest that a gene called CSL, which binds to Notch, may prevent normal cell development. By inserting this gene into normal and leukaemic cells in culture, we hope to gain an understanding as to how, and why, this might be the case. We will also explore the possibility that other members of the Notch family are involved in leukaemia development.
We are particularly interested in addressing whether Notch-1 mutations occur in all age groups of patients with T-ALL and whether they are involved in other forms of acute leukaemia apart from T-ALL. This is particularly timely as drugs that inhibit Notch are in development for the treatment of T-ALL.
.

The Notch pathway, pivotal in directing cell fate in embryonic development, is now recognised as having an important role in regulation of haemopoiesis. Notch directs the transition from common lymphoid stem cell to committed T-cell progenitor, and has recently been shown to regulate the stem cell niche, affect myeloid differentiation, and control haemopoietic stem cell self-renewal, all of which are dysregulated in leukaemogenesis. It is a membrane-bound receptor; on ligand binding its intracellular portion is cleaved and translocates to the nucleus to bind the transcription factor CSL. It has recently been discovered that >50% of paediatric/adolescent patients with T-cell acute lymphoblastic leukaemia (T-ALL) have activating mutations in the Notch-1 receptor in either the heterodimerisation or PEST domain, and sometimes in both domains. Notch-3, which shares many features with Notch-1, including its ability to produce a T-cell leukaemia on transfection into mice, is a likely target gene in those cases of T-ALL without a Notch-1 mutation. We hypothesise that mutations of the Notch pathway may be found in acute myeloid leukaemia (AML) patients where there is aberrant T-cell expression. Infants with leukaemia often have mixed lineage surface antigen expression and this rare group of patients also warrants detailed analysis. Additionally, we have found that ˜50% of patients with AML have a previously undescribed isoform of CSL where exon 10 is truncated (termed CSL-TREX). When AML blasts can be induced to differentiate in vitro, CSL-TREX is down-regulated, thus raising the possibility that it contributes to maintenance of the undifferentiated state in vivo.

Samples from a cohort of infant leukaemia patients, accessed from the UCL Institute of Child Health DNA bank, will be screened for Notch-1 and Notch-3 mutations by heteroduplex analysis, and positive samples sequenced. Notch-1 will be studied in 50 adult AML patients with aberrant T-cell expression and compared with 50 patients without these markers, as identified from the MRC DNA bank. In order to explore the possibility of autocrine stimulation, the Notch ligand Jagged-1, Notch-1 and the downstream molecule HES-1, will be quantified by real-time RT-PCR of 200 AML RNA samples. We will use a single cell, Notch-sensitive, GFP-reporter system to analyse Notch activation in de novo AML cells. Finally, we will further characterise the function of CSL-TREX by transfecting CD34+ cells using a GFP-expressing lentiviral vector. These studies are particularly timely as -secretase inhibitors that inhibit Notch function are already in phase I/II trials in childhood T-ALL.