Role of Early B Cell Factor-1 (Ebf1) in replication initiation of normal B cells and in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL).

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B cells are white blood cells that produce antibodies to eliminate infections. These cells develop in the bone marrow from progenitor cells, and they grow, multiply and die under controlled circumstances. In Acute Lymphoid Leukaemia, the fine balance between these processes becomes unstable.Early B Cell Factor 1(Ebf1) is a gene that is crucial for the development of B cells, and mutations in this gene are found in a high proportion of Acute Lymphocytic Leukaemia (ALL) patients.By genetic inactivation of this gene in mice, we have found that B cells which do not express Ebf1 were unable to initiate DNA-replication. To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Licensed origins, potential starting points of replication are in tenfold excess over the number of origins normally used, to make sure that if some origins fail to fire, another nearby origin takes over to ensure that the entire length of DNA is duplicated. We hypothesise that in pre-leukaemic Ebf1-mutant cells the DNA-replication licensing process is disturbed, but the surveillance machinery, which is called a replication licensing checkpoint, prevents the DNA-replication process to start until cells accumulate the sufficient number of licensed origins. If the surveillance mechanism is not working, the cells can start dividing before the duplication of their DNA is finished. Fortunately, cells that contain unreplicated DNA are recognised and eliminated by a subsequent checkpoint in the S or G2 phase of the cell cycle, before the onset of cell-division. In some leukaemic cells even this second safety mechanism is destroyed, and we believe that in normal progenitor B cells the known oncogen, c-myb controls this process. The current proposal aims to find out how exactly Ebf1 instructs the B cells to start to replicate their DNA. In our model, loss of Ebf1 represents the first stage of carcinogenesis, loss of the replication licensing checkpoint represents the second stage. It is an important therapeutic possibility to inhibit the replication licensing process in checkpoint-deficient cancer cells. Normal cells respond to such inhibitors with reversible growth arrest, but checkpoint-deficient cancer cells proceed with replication and die due to the activity of the S/G2 checkpoint. The Cdc7 serine/threonine kinase plays an essential role in origin licensing, and inhibition of Cdc7 has been demonstrated to selectively induce cell death in malignant cells. We demonstrated that Cdc7 inhibition mimicks the effect of Ebf1-mutagenesis in a mouse model of ALL. We were able to categorise our murine leukaemic cells to two distinct types based on their response to Cdc7 inhibition. Interestingly, the cell cycle arrest versus cell death response to Cdc7 inhibition predicted a similar response to the loss of Ebf1 in ALL cells derived from our Ebf1 conditional knockout mouse, in which we could delete the Ebf1 gene in an inducible manner. Furthermore, we observed the same two response types upon treatment of cells with Gleevec, the famous kinase inhibitor that dramatically improved the clinical outcome of BCR-ABL1 (or Philadelphia chromosome) positive leukaemias. The proposed research aims at the better understanding how the replication licensing checkpoint works in normal B cells, why it is disturbed in ALL, and finally, we plan to discover biomarkers, measurable parameters of leukaemia cells that would predict the efficacy of this drug in human ALL cells, leading to future personalised therapy in leukaemia patients.