Understanding the requirement of WNT pathway activation

Understanding the requirement of WNT pathway activation in the initiation and progression of the pituitary tumour craniopharyngioma

Adamantinomatous craniopharyngioma (ACP) is a clinically aggressive tumour in children and adults for which there are not curative treatments. Patients suffer a high degree of morbidity (e.g. blindness, severe obesity, multiple hormonal deficits) leading to poor quality of life and increased mortality in long-term follow-up. Research from the applicants’ lab have provided important insights into the pathogenesis of ACP and identified potential pathways that can be targeted with specific inhibitors [1].

Using unique genetically modified mouse models (GEMMS), we have demonstrated that mutations in CTNNB1 (encoding beta-catenin) are drivers of tumourigenesis in the mouse models and in human patients [2,3]. These mutations result in the over-activation of the WNT pathway leading to tumour initiation. However, we do not know whether elevated WNT pathway is required just for tumour initiation or in addition is necessary for further tumour growth and progression. This is important not only to understand better ACP pathogenesis, but also to provide a rationale for the use of WNT pathway inhibitors, which are currently being tested in clinical trials [4].

This PhD project will address fundamental questions on the function of the WNT pathway in ACP by using a multidisciplinary approach, combining mouse genetics with molecular and cellular biology. Ultimately, this information will provide proof-of-principle for the initiation of clinical trials.

Aims/Objectives:
The PhD includes three Aims:
Aim 1. The student will generate a new Col1a1tm1(tetO-Ctnnb1*-mCherry) mouse line as described 5. This line will express, in a tetracyclin-inducible manner, oncogenic beta-catenin (i.e. CTNNB1 gene carrying the most common mutations identified in human ACP) as well as mCherry. This line will be crossed to our Sox2CreERT2 mouse line 2 and the Rosa26-CAGs-LSL-rtTA3 line (available at the ICH and the JAX, stock No: 029617).
Aim 2. The new mouse line will allow the activation of oncogenic beta-catenin in SOX2 stem cells only when mice are exposed to tetracycline (Tet), but expression will be shut down upon Tet withdrawal. This will allow the student to assess when and for how long is elevated WNT pathway activation required for tumourigenesis.  The student will evaluate tumour burden and will use state-of-the-art approaches such as scRNA-Seq and RNA-Scope to characterise the phenotypes obtained. 
Aim 3. To further explore the potential WNT pathway inhibition against ACP, the student will use several drugs, which are currently being tested in ongoing clinical trials. This is an intensive area of research and through the oncologists at GOSH, (e.g. Darren Hargrave) we have initiated contacts with the industry to test some inhibitors.

Methods:
The student will combine mouse genetics (ES cell targeting using CRISPR/Cas9 technology), molecular (scRNA-Seq, RNA-Scope, immunostaining)  and cellular (cell culture) approaches to dissect the requirement of WNT pathway activation in the genesis of ACP and to explore novel targeted treatments in preclinical research.

Timeline:
The primary supervisor is the academic lead of UCL Transgenics, a core technology platform that among other approaches specialises in the generation of new mouse models. The PhD student will be supported by this infrastructure and importantly by the personnel in the facility. This will guarantee the generation of the mouse line in the first year of the PhD. All the other mouse lines are available at UCL. It is anticipated that Aim 1 will be achieved before the upgrade (beginning of Year 1). Aim 2 and Aim 3 will be finished in the following 16-18 months, leaving 4-6 months for the thesis write up.

References:
1.  Muller, H. L., Merchant, T. E., Puget, S. & Martinez-Barbera, J. P. New outlook on the diagnosis, treatment and follow-up of childhood-onset craniopharyngioma. Nat Rev Endocrinol 13, 299-312, doi:10.1038/nrendo.2016.217 (2017).
2.  Andoniadou, C. L. et al. Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell 13, 433-445, doi:10.1016/j.stem.2013.07.004 (2013).
3.  Gaston-Massuet, C. et al. Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans. Proc. Natl. Acad. Sci. U. S. A. 108, 11482-11487, doi:10.1073/pnas.1101553108 (2011).
4.  Krishnamurthy, N. & Kurzrock, R. Targeting the Wnt/beta-catenin pathway in cancer: Update on effectors and inhibitors. Cancer Treat. Rev. 62, 50-60, doi:10.1016/j.ctrv.2017.11.002 (2018).
5.  Beard, C., Hochedlinger, K., Plath, K., Wutz, A. & Jaenisch, R. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44, 23-28, doi:10.1002/gene.20180 (2006).