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).
Lead Research Organisation:
University of Leicester
Department Name: Biochemistry
Abstract
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 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.
Technical Summary
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.
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.
Planned Impact
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
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
People |
ORCID iD |
Ildiko Gyory (Principal Investigator) | |
Matthew Blades (Researcher) |
Publications
Tinterri A
(2018)
Active intermixing of indirect and direct neurons builds the striatal mosaic.
in Nature communications
Description | participation in review committee of applications to the Molecular Biology panel of the National Research, Development and Innovation Office of Hungary |
Geographic Reach | Europe |
Policy Influence Type | Membership of a guideline committee |
Impact | Involving scientists from other EU countries ensures that the review system is not corrupted and internationally good standards are kept. |
URL | http://nkfih.gov.hu/kutatas-fejlesztes |
Description | Development Fund |
Amount | £5,000 (GBP) |
Organisation | Hope Against Cancer (Rutland and Leicestershire) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 11/2014 |
End | 11/2015 |
Description | MRC IMPACT Doctoral Training Studentship |
Amount | £75,000 (GBP) |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2017 |
End | 03/2021 |
Description | Midlands Integrative Biosciences Training Partnership (MIBTP) is a BBSRC-funded doctoral training partnership |
Amount | £0 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2014 |
End | 07/2018 |
Description | Midlands Integrative Biosciences Training Partnership (MIBTP) is a BBSRC-funded doctoral training partnership |
Amount | £0 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2015 |
End | 06/2019 |
Description | Midlands Integrative Biosciences Training Partnership (MIBTP) is a BBSRC-funded doctoral training partnership |
Amount | £75,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2017 |
End | 08/2021 |
Description | Research Grant |
Amount | £15,000 (GBP) |
Funding ID | RG130495 |
Organisation | The Royal Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2014 |
End | 02/2015 |
Title | $Expansion of GFP+ cells as measure of cellular fittness |
Description | In order to examine which Ebf1 domain is pivotal for the well-being of pro-B cells, novel cell lines were generated with gene replacement involving the transduction of A-MuLV-transformed pro-B cells from Ebf1fl/flRERTCre mice with EBF1wt or specific domain deleted EBF (EBF1?-DBD, EBF1?-IPT, EBF1?-HLH and EBF1?-TAD) retroviruses and the endogenous Ebf1 alleles were inactivated by the treatment of cells with 4-OHT. The success of the gene replacement approach depends on the ability of the domain-deleted Ebf1 proteins to substitute the function of Ebf1 in survival and proliferation. The EBF1wt, EBF1?-IPT, EBF1?-HLH and EBF1?-TAD proteins fully substituted the endogenous Ebf1 protein, evidenced by the substantial increase of GFP positive cells (Figure 5B, S2A) and stable survival of the cell lines. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2015 |
Provided To Others? | Yes |
Impact | manuscript in submission |
Description | B-lymphoid malignancies |
Organisation | University of Leicester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | mouse models of B-lymphoid malignancies and expertise in in vitro lymphocyte work |
Collaborator Contribution | drug development work and translation of basic research into clinical setting |
Impact | Both labs are working with mouse models of lymphoma. We share reagents and expertise and two undergraduate students are working under joint supervision of the two labs on projrect that are under the scope of the MRC grant |
Start Year | 2013 |
Description | Regulation of EBF1 on B cell development after bone marrow transplantation |
Organisation | Sun Yat-Sen University |
Country | China |
Sector | Academic/University |
PI Contribution | Co-investigator on the partner's grant application from the National Natural Science Foundation of China in 2017 to study "The regulation of EBF1 on B cell differentiation and development and the mechanism involved in the mesenchymal stem cell treatment of chronic graft-versus-host disease." |
Collaborator Contribution | Adoptive transfer of third-party mesenchymal stromal cells (MSCs) is a novel method of treating chronic graft-versus-host disease (GVHD). The laboratory of the co-applicant has discovered that differences in the B cell numbers and subtypes correlated with the therapeutic outcome. From the perspective of basic B cell biology, these CD27+ memory and pre-germinal centre B lymphocytes represent an unusual line of development, and their expansion is instructed by the transplanted MSCs. This project aims to understand the molecular mechanisms of how MSCs influence B cell development, and to identify the precursors. |
Impact | This is a new partnership that has recently been established based on the intersecting findings of our research about basic B cell developmental biology and Dr. Peng's translational research about Graft Versus Host disease. |
Start Year | 2017 |
Description | p53 in B lymphocyte |
Organisation | University of Leicester |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Research on how the p53 pathway is affected by B-cell specific signals |
Collaborator Contribution | Research on how B-lymphocytes respond differently upon p53 activation |
Impact | The partnership has just recently formed. The Macip and Gyory lab share weekly lab-meetings to discuss topics that are interesting for both parties and to share experiments and materials |
Start Year | 2013 |
Description | Genes and Cancer Meeting 2017 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | This was the 32nd edition of the Genes & Cancer Meeting, the longest running meeting in the UK, for the presentation of new advances in the field of international cancer research, from bench to bedside. Large scale event aiming to professional and international audiences. |
Year(s) Of Engagement Activity | 2017 |
URL | http://genesandcancer.org.uk |
Description | MIBTP Masterclass |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | MIBTP master-classes are aimed at broadening the doctoral students horizon in research techniques |
Year(s) Of Engagement Activity | 2015,2016 |