Finding therapeutic targets in FLT3-ITD AML using a systems biology approach

Leukaemia is a blood cell cancer that arises when stem cells or immature blood cells are hit by a series of mutations in their DNA. The consequence is that the cell starts to activate genes that are not normally active, or genes are altered to make abnormal proteins, or no protein at all. If such a protein is required to switch other genes on or off, the consequences can be devastating. The reason for this is that the finely balanced order in which genes are switched on or off during blood cell development is now disturbed. In the early stages, a single mutation may have little effect, and many apparently normal people already carry some mutations in their blood cells. However, additional mutations create a domino effect. First, some target genes are de-regulated by the mutations. This can lead to stem cells that grow more than they should, but are otherwise quite normal and still can form normal blood cells. Over time additional changes occur that tip the balance from a cell that grows a bit too much, to cells where blood cell development grinds to a halt and the cells become malignant. Such cells do not develop into normal blood cells, but form leukemic cells that keep growing and growing until they finally take over the body.

Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults. Despite improvements in supportive care, outcome typically remains poor for older AML patients. It has long been known that AML cannot be classified as just one disease but is highly heterogeneous, involving different genetic mutations and highly variable clinical outcomes. The FLT3-ITD mutation is a growth-promoting mutation, and one of the mutations that has the most devastating effects. It occurs in about 25% of all cases, and generates a continuously active protein that cannot be switched off, which tells cells to grow indefinitely. The clinical prognosis of having such a mutation is dire, and treatment with drugs targeting the FLT3-ITD protein soon results in the development of drug resistance and relapse. The Bonifer/Cockerill group has recently embarked on a series of experiments which highlighted how gene regulation is altered in AML with FLT3-ITD and deviates from normal cells. This was made possible by modern technology that looks at many genes simultaneously. We have uncovered a network of genes which are likely to be essential for the development and maintenance of FLT3-ITD AML. These include the transcriptional regulators RUNX1 and AP-1 which control the abnormal expression of FLT3-ITD AML-specific proteins. We have now teamed up with the Heidenreich lab who developed an in vivo model of human FLT3-ITD AML and the lab of John Bushweller from the University of Virginia who has developed novel drugs that target RUNX1 directly. Our proposed work will build on our results and is designed to (i) identify new targets for therapy, (ii) understand which genes are affected by different drug and (iii) use optimized drugs in mouse models of AMLs to prepare the stage to test these novel molecules in a clinical trial.

In this work we will use inhibitory RNA molecules to block the production of proteins that are aberrantly expressed in FLT3-ITD AML. We will use this screen to identify which of the abnormally expressed genes are vital to the growth of these AML cells. Once we have identified genes and pathways that control the gene regulatory network we will use specific reagents and chemical inhibitors to block these points in the AML network, and block leukaemia development. These will include (a) a drug that than specifically block the binding of the DNA-binding transcriptional regulator RUNX1, (b) A shortened version of the FOS protein which acts as a dominantly acting repressor of all members of the AP-1 family of transcriptional regulators, and (3) combinations of clinically approved inhibitors of FLT3-ITD and MAPK signalling, which we predict will be more effective than therapies using single agents.

Acute myeloid leukaemia (AML) is caused by mutations interfering with hematopoietic differentiation and mutations conveying a proliferative advantage. FLT3-ITD mutations are the most common growth-promoting mutations in AML. The prognosis of FLT3-ITD AML is extremely poor and new therapies are urgently needed. For this we need to obtain systems-level information on the targets affected by drugs and mechanistic information on how drugs work. We have extensively profiled FLT3-ITD AML using multi-omics approaches and uncovered a common set of FLT3-ITD AML-specific deregulated genes. We identified the core FLT3-ITD-specific deregulated transcriptional network and pathways. Central to this network was FLT3-ITD signalling to MAPK, AP-1 and RUNX1. In collaboration with John Bushweller in the USA, we showed that small molecule inhibitors of RUNX1 can suppress the FLT3-ITD network. We also established conditions to propagate primary FLT3-ITD cells both in vitro and in vivo.
In this proposal we will build on this work to define the role of such genes and pathways with respect to FLT3-ITD AML development and maintenance.
(1) We will use an shRNA-based drop-out screen targeting the complete list of FLT3-ITD-AML-specific genes. We will use this to identify genes essential for tumour formation in a FLT3-ITD xenograft model, or for colony formation using an in vitro model.
(2) We will validate the role of specific signalling pathways, transcription factors and other genes using patient-derived FLT3-ITD AML cells. We will target these parts of the FLT3-ITD network using (a) shRNAs identified via the shRNA screen, (b) RUNX1 inhibitors, and (c) A dominant negates FOS which acts as a broad inhibitor of AP-1.
(3) We will also use drugs to target essential pathways in FLT3-ITD AML. We will test combinatorial therapy of FLT3 inhibitors plus different MAPK inhibitors.
(4) We will reveal new therapeutic targets by identifying pathways used by FLT3-ITD AML to develop resistance.

Who will benefit from this research:
Our work will have an impact not only on our immediate research field but also far beyond. Currently the scientific community has embarked on a quest to not just study single genes within single cells, but to examine biological phenomena in a systems-wide fashion using high-throughput methodologies. However, a biological system is an integrated unit that functions on multiple levels and employs multiple feedback mechanisms to maintain its identity. It is these feedback mechanisms which are responsible for the reprogramming of transcriptional networks in a cancer setting where important regulators are either defect or aberrantly active.
If successful, our work will benefit a number of diverse research fields. This includes
(i) myeloid biology, because so far the relevance of aberrant signalling for the regulation of differentiation is not well understood
(ii) epigenetics research, because it will benefit from understanding how aberrant signalling feeds back on the epigenome
(iii) mathematical modelling and bioinformatics research, because our data will provide ample opportunity for mathematical modelling and developing novel methods for data integration.
(iv) leukaemia research, because our studies of identifying how aberrant signalling is linked with differentiation potential will be of utmost importance for the understanding of tumour cell behaviour.
(v) drug development as our global studies of resistance acquisition will gather profound insights of the feedback mechanisms that AML cells develop to survive which will led to the identification of additional drug targets.
(vi) clinical research as we will test a novel class of drugs

How will they benefit from this research?
(i) We will make our system-wide datasets and network models publicly available.
(ii) We will generate data that will be highly relevant to scientists studying other developmental/differentiation pathways both in academia and industry.
(iii) One significant potential outcome of our work is the identification of how aberrant signalling shapes differentiation which may identify new drug targets. We will make our expertise available to members from industry and academia who wish to explore this possibility.
(iv) Through our international collaborations there will be a significant knowledge transfer into the UK
(v) Our work will enhance the skills base in the UK. Future advances in biology and medicine will depend on building a skills base consisting of researchers which will be capable of thinking both in molecular terms as well as in system-wide terms, and researchers working on this grant will be exposed to the forefront of research in this field.
(vi) Along the same lines, we are also hoping to attract domestic and international students into this area. Such students will be encouraged to participate in our programme and work with the data we are generating.
vii) Strong therapeutic candidates will be discussed with the Newcastle Drug Discovery Unit for potential drug development activities. The unit has established a strategic partnership with ASTEX, which will facilitate commercialisation and further development of promising targets and lead structures.