Establishing the mechanism for RUVBL2 essentiality in acute myeloid leukaemia

Despite remarkable advances in the treatment of common leukaemia in children, some types of this disease, such as acute myeloid leukaemia (AML), are still difficult to treat. Current therapy fails to cure 1 in 3 children diagnosed with AML, and over half of adult patients, indicating an urgent need for new and improved treatments. There is general consensus in leukaemia research that advances likely to result in such new therapies rely on achieving a greater understanding of the molecular basis of the disease. Our group studies how cancer genes hijack normal cellular pathways to promote conversion of normal blood cells into leukaemia. We and others have shown that the molecule c-MYB mediates cancer gene function in most AML types, ensuring that AML cells continuously divide, producing copies of themselves, and that they do not differentiate into mature blood cells. In AML patients, this results in the replacement of normal blood cells with non-functional leukaemia cells, leading to catastrophic deficiencies in tissue oxygen delivery, poor blood clotting and susceptibility to infection. However, targeting c-MYB itself is problematical, mainly due to its requirement for normal blood cell development. We recently uncovered a pathway operating in AML cells, in which the protein RUVBL2 controls the activity of c-MYB, ensuring it blocks differentiation of AML cells. Although inhibition of RUVBL2 resulted in the death of AML cells, it had little impact on normal blood cell development. This suggests that therapies targeting this protein may be effective and relatively non-toxic. This project aims to characterise how RUVBL2 controls c-MYB and to discover what parts of RUVBL2 are necessary for this control. We will then examine the effects of interfering with control of c-MYB function by RUVBL2 on AML cells using experimental models of the disease. We will use the results from this project to determine how effective new therapies that block RUVBL2 function are likely to be and envisage using this information for future development of these therapies in AML.

Research in our groups is focussed on understanding the molecular basis of acute leukaemia. We reported previously that the AAA+ ATPase RUVBL2 is essential for acute myeloid leukaemia (AML) survival. In this proposal we present further evidence for the selective requirement of RUVBL2 in AML, but not normal haematopoietic progenitors. We demonstrate that RUVBL2 binds to the transcription factor c-MYB and regulates its activity, ensuring repression of myeloid differentiation-associated c-MYB target genes. c-MYB is known to play an essential role in multiple subtypes of AML. The mechanism underlying the control of c-MYB activity by RUVBL2 in AML is unclear and the subject of the current proposal. We propose to determine how RUVBL2 controls c-MYB activity, by establishing the impact of RUVBL2 on co-repressor and co-activator complexes recruited to c-MYB and to c-MYB target genes, and examine changes to this recruitment and to the target gene chromatin landscape following RUVBL2 silencing. We will generate deletion and point mutations to define which regions and amino acid residues of RUVBL2 are necessary for c-MYB binding and its regulation, and examine the effects of inhibitory RUVBL2 mutants on essential leukaemia-specific transcriptional networks, and on AML survival and progression in experimental models of the disease. Further analysis of our data also demonstrated changes in E2F1 target gene expression in AML cells following RUVBL2 silencing. Following on from a recent study reporting RUVBL2 binding to E2F1 and regulation of its transcriptional activity in hepatocellular carcinoma, we will examine interactions of RUVBL2 with E2F1 in AML cells and the impact of RUVBL2 on target gene binding by E2F1. We envisage that this project will provide crucial information on how RUVBL2 contributes to the establishment of aberrant leukaemia-associated transcription factor networks and will facilitate future drug development initiatives.

1. In the long-term, the proposed research will benefit paediatric and adult patients diagnosed with AML, and their families. This work will identify regions and amino acid residues of RUVBL2 that are involved in mediating aberrant regulation of c-MYB activity, necessary for leukaemia progression, and determine the potential therapeutic efficacy of inhibiting this regulation using patient-derived AML models. Development of novel, targeted and less toxic therapies for AML is a clinically unmet need. Our data indicate that normal haematopoiesis is less susceptible to RUVBL2 inhibition than AML, suggesting the existence of a therapeutic window for RUVBL2 targeting in this disease.

The data from this research will enable us to propose new drug development initiatives in future studies, specifically targeting RUVBL2-mediated regulation of c-MYB, such as applications to the CRUK Small Molecule Drug Discovery Project Awards. We have previously discussed the potential of RUVBL2 targeting approaches with the Drug Discovery Unit, CRUK Manchester Institute. This project addresses many of the mechanistic details that will facilitate development of such approaches.

This project will be performed in laboratories that are embedded in a translational framework. Close interaction with clinical colleagues involved in treating AML patients will ensure the full exploitation of this research. Dr Marc Mansour (co-investigator on this proposal) is a clinician-scientist based at the UCL Cancer Institute, currently treating adult AML patients at the University College London Hospital (UCLH). Dr Owen Williams (principal investigator) is co-located with senior consultant paediatric haematologists treating AML patients at Great Ormond Street Hospital (GOSH) and engages in both formal and informal meetings discussing translational aspects and potential of current research.

2. It is likely that this project will generate intellectual property and that biotechnology and pharmaceutical companies will be interested in the outcomes of this research. As well as studying the basic mechanisms of oncogene function in leukaemia, our long-term aim is to develop new drugs with which to treat AML. This project will provide crucial mechanistic details that will enable us to apply for future drug development funding. It is highly likely that leukaemia susceptibility to RUVBL2/c-MYB inhibition is not specific to paediatric AML but will be equally applicable to adult disease. This significantly increases the numbers of patients that can be enrolled into clinical trials and the patient population that will benefit from development of new drugs. This is an important consideration in the cost-benefit analyses of pharmaceutical companies and increases the likelihood that the 'patient market' will be of sufficient size to recover drug development and clinical trial costs.

3. The research will also benefit organisations such as charities that are committed to funding medical research to improve healthcare provision for leukaemia and cancer patients. Charities such as GOSHCC, CWCUK and Bloodwise produce general information on different types of leukaemia and lay summaries of current research into new therapies, in the form of leaflets and information on their websites, for patients, their families and the general public. This contributes to increased public awareness of leukaemia, current challenges in its treatment and the contribution of scientific research to meeting those challenges. These charities may find the results of this project useful to include in their material.