Close
Supervisors: Dr Andrew Stoker
Background
Neuroblastoma is an extracranial solid tumour in children with a poor prognosis for those patients with high risk disease [1]. Although there are a number of oncogene targets for this disease, including N-Myc [2] and ALK kinase [3], the ability to target these proteins and their signaling pathways remains only partially successful. Cell signaling is regulated at a high level by protein tyrosine phosphorylation events, controlled by kinases and phosphatases (PTPs), both of which can be oncogenic [4]. The ability to improve the drug targeting of both of these enzyme classes, including combination approaches, may be critical for optimal treatments in future. We have recently completed a screen of one subfamily of PTPs, the dual specificity phosphatases, DUSPs, addressing their potential role in promoting cellular hallmarks of cancer in neuroblastoma cells. We identified two DUSPs with potential “oncogene-like” characteristics and this project aims to expand on this knowledge and to get us closer to understanding whether or not these DUSP enzymes, or their downstream pathways, represent viable drug targets in this cancer. Our recent research (E.Thompson thesis, unpublished) has identified DUSP6 as an enzyme that appears to operate downstream of the oncoprotein ALK. We now need to understand DUSP6’s novel function in terms of its unknown biochemical targets. A second enzyme DUSP3 has been implicated in other cancers as a potential oncogene, and we have discovered that loss of DUSP3 function in neuroblastoma cells can reduce their uncontrolled proliferation and stemness. Again, we aim here to investigate how DUSP3 operates in neuroblastoma cells and to investigate if this enzyme or its downstream pathways can be targeted with inhibitors. With both DUSP3 and DUSP6, we are also interested to uncover novel drug targeting approaches that are effective in combination with ALK or other neuroblastoma-specific oncogene pathways.
Objectives
- We will define the cellular consequences of loss-of-function and gain-of-function in both DUSP6 and DUSP3 enzymes in a range of neuroblastoma cell lines
- We will further define how these DUSPs interact biochemically with known oncogenic pathways in neuroblastoma cells
- We will locate existing drugs that target the effectors of DUSP and are effective in combination treatments alongside inhibition of other known oncoproteins.
- Using xenografted tumours, we will determine if loss-of-function in either DUSP6 or DUSP3 pathways reduces tumour formation
Methods
- Using CRISPR/Cas9 technology the student will establish neuroblastoma cell lines with DUSP3 gene inactivation (related DUSP6-targeted cells already exist). Inducible protein degradation systems (e.g. auxin-inducible degron (AID)) will be used to tag endogenous DUSP genes. This will permit the temporal degradation of the proteins so that acute signaling dysfunction can be defined and understood [5].
- Using approaches such as immunoblotting, phosphoantibody arrays and phosphoproteomics, these loss-of-function model cells will be characterised for their changes in intracellular signalling and in particular signaling through ALK, MTOR and N-Myc pathways. Cells will also be characterised for changes in proliferation, stemness and invasiveness.
- Inhibitors that are predicted to block pathways downstream of these DUSP enzymes will be combined with inhibitors of ALK, MTOR or N-myc, to examine drug synergy.
- Cell with mutations in DUSP genes will be tested for their reduced ability to grow as xenografts in vivo. Furthermore, the combination drug treatments developed above will be used in vivo to assess enhancement in anti-tumour efficacy.
References
1. Brodeur, G.M. Nat Rev Cancer, 2003. 3(3): p. 203-16.
2. Huang, M. and W.A. Weiss. Cold Spring Harb Perspect Med, 2013. 3(10): p. a014415.
3. George, R.E., et al. Nature, 2008. 455(7215): p. 975-8.
4. Julien, S.G., et al.. Nature reviews. Cancer, 2011. 11(1): p. 35-49.
