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).

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.

Early B cell factor 1 (Ebf1) is a DNA-binding protein with a pivotal function in the specification of the B lymphocyte lineage. Mutations in the EBF1 gene or deregulated EBF1 expression are found in a high proportion of BCP-ALL cases, and a direct role of Ebf1 in leukaemogenesis is supported by several mouse models of ALL.
By conditional gene inactivation, we have found that Ebf1 is required for proliferation and survival of B cells at virtually every stage of their development. Loss of Ebf1-expression results in a phenotype that highly resembles activation of the replication licensing checkpoint.
This cell cycle checkpoint represents a physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. DNA-damage or depletion of replication licensing factors (e.g. Cdc6 or Cdc7) result in activation of this novel G1 checkpoint that prevents S phase progression until the sufficient number of replication-competent origins is achieved. Cancer cells with a deficient checkpoint progress through S-phase, but are eliminated by a subsequent intra-S or G2 checkpoint. Ebf1-deleted transformed cells die without arrest, similarly to tumour cells depleted of Cdc6 or Cdc7 by siRNA. We have found that transformed, but not primary pro-B cells survive upon exogenous c-myb expression (Gyory, Boller et al. 2012). Thus, the lethal S-phase in transformed cells is overcome by overexpression of c-myb or b-myb, which are known transcriptional inducers of G2/M genes.
The aim of this proposal is to elucidate the exact role of Ebf1 in replication initiation, to identify the factor(s) that are responsible for the maintenance of the replication licensing checkpoint in normal B lymphocytes and that are potentially deregulated in human ALL. We are planning to investigate the therapeutic importance of replication licensing inhibition in this disease.

1. Academic Impact
The proposed research will gain input from and will strengthen the potential of the Cancer Theme within the College of Medicine, Biological Sciences and Psychology at the University of Leicester. The proposed translational work will complement ongoing drug development work and with the support of Professor Dyer, could be taken into a clinical setting.
2. Commercial / Industrial beneficiaries -
Inhibition of Cdc7 has been demonstrated to selectively induce cell death in tumours versus induce a reversible cell cycle arrest in non-malignant cells. The current proposal aims to define the biological properties of different subtypes of Acute Lymphoid Leukaemia in the context of their response to Cdc7 inhibition. The Cdc7 inhibitors are recognised as emerging and potent drugs within the pharmaceutical industry, and our preliminary data indicate that cellular response to these drugs may predicts sensitivity to other classes of therapeutic compounds. The University of Leicester has a well-established 'Enterprise & Business Development' team and an embedded unit ('The Biobator'), dedicated to exploitation of findings generated in biomedical research. Outputs from the project will be used by BIOBATOR to establish partnerships with industrial collaborators of the leukaemia therapy field.
3. Societal beneficiaries-
a. We are planning to translate our findings that are emerging from an animal model system to biomarker discovery in human leukaemia. Our longer term goal is to personalise chemotherapy and thereby reduce costs, risks and side-effects associated with the treatment of leukaemia patients while increasing therapeutic benefit. From compound discovery to clinically useful drugs the developmental path takes decades. The Cdc7 inhibitors, a promising new group of anti-cancer drugs have reached phase I and II clinical trials already i certain types of tumours. The proposed research will help defining the area of potential use which will benefit patients in a relatively near future of 5 years.
b. The anticipated research outcomes of the current proposal will have a high impact on the the lab as a teaching environment. For many undergraduate and graduate students this project will be a critical encounter with scientific thinking, data generation and management and cancer research in general