MITF AND MELANOMA RESISTANCE TO TARGETED THERAPIES

Abstract

Melanoma is a highly aggressive form of skin cancer, in which MAPK pathway deregulation is common. Because of this, MAPK pathway inhibitors are used in the clinic to treat melanoma; complete or partial tumour regression is observed in most patients, however resistance is inevitable. Hyperactivation of the PI3K pathway and re-activation of the MAPK pathway have been observed in resistant melanoma. Additionally, changes in expression and activity of the lineage-specific transcription factor, MITF, are also observed in resistant melanoma. MITF is a crucial regulator of survival and differentiation genes in melanoma. A fraction of relapsed tumours express high levels of MITF, however the majority of relapsed tumours (approximately 50%) show reduced MITF expression. MITF-null resistant cells that are instead driven by WNT5A signalling are completely unresponsive to MAPK pathway inhibitors due to rewired cell signalling. These tumours have been correlated with an increased activation of EGFR and therefore enhanced MAPK pathway signalling, leading to resistance. In contrast, WNT5A has been shown to trigger PI3K signalling. In this project, we aimed to identify whether PI3K pathway hyperactivation and hyperactivation NRAS mutation lead to a specific melanoma cell-fate; specifically whether they determine whether MITF expression is increased or decreased in relapsed tumours. We used small molecule inhibitors of PI3K and MAPK pathways to show that under normal conditions (not in a resistance setting) MAPK signalling positively contributes to MITF expression, whereas PI3K signalling does not appear to affect MITF expression in A375 or WM266-4 cells. Next to identify whether hyperactivation, we generated isogenic cell lines expressing hyperactive NRAS or PI3K constructs, however the cell lines produced could not fully recapitulate what is observed in drug resistant cells. We therefore exploited the M249 model of resistant melanoma; these resistant cells express an activating NRAS mutation and are therefore able to re-activate the MAPK pathway in the presence of a BRAF inhibitor. We have treated these cells with the MEK inhibitor, MEK162, and begun to understand how MITF expression and its target genes are regulated. Furthermore, we have identified that long-term treatment of M249 cells with low doses of MEK and BRAF inhibitors (Trametinib and Dabrafenib) result in loss of MITF expression. We hope that further analysis of these cells will enhance our understanding of the control of MITF expression during resistance to MAPK pathway inhibition.

Date of Award	1 Aug 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Claudia Wellbrock (Supervisor) & Andrew Sharrocks (Supervisor)