Characterisation of MYB and TRKB as candidate targets in CYLD cutaneous syndrome

Abstract

CYLD cutaneous syndrome is a rare, autosomal dominant inherited disease in which an individual is predisposed to developing multiple cutaneous tumours; namely cylindromas, spiradenomas and trichoepitheliomas, due to germline mutations in the tumour suppressor gene CYLD. However at present there are no curative treatments other than repetitive surgery to control tumour burden. Therefore, an overarching theme of this thesis was to identify potential therapeutic targets that could possibly translate into treatment strategies in the clinic. The first strategic target, c-MYB, was explored, as overexpression of c-MYB was previously shown in sporadic cylindromas due to the presence of the fusion protein MYB-NFIB. It was demonstrated that inherited cylindromas did not express the MYBNFIB fusion protein. However, using immunohistochemistry c-MYB was shown to be overexpressed in inherited tumours and that gene silencing of MYB caused a reduction in cell viability. A novel finding, transcriptomic and protein analysis also revealed that keratinocytes expressed the alternate MYB 9B isoform. A second strategic target, Trk, was explored using RNA-seq of CYLD defective tumours. Exploration of Trk signalling in these tumour cells found that the truncated TrkB.T1 isoform of the TrkB receptor is overexpressed in the tumours, alongside the receptors cognate ligand BDNF. Overexpression of TrkB.T1 in the HaCaT, keratinocyte cell line, indicated that TrkB.T1 increased cell survival. BDNF stimulation of TrkB.T1 overexpressing cells caused Stat3 phosphorylation, and in primary cylindroma tumour cells BDNF stimulation increased the nuclear localisation of Stat3. Finally, a three-dimensional spheroid cell culture method was established and characterised using immunofluorescence, as 3D in vitro models better reflect the in vivo environment. The cultures were found to have an expression pattern similar to their in vivo counterparts and were shown to be sensitive to inhibitory Stat3 targeting, supporting the translational relevance of this work in the hope of bringing targeted therapies for CYLD cutaneous syndrome to the clinic.