Exploring the regulation of MITF in the context of response to MAP-kinase targeted therapy

Abstract

This thesis brings together six peer-reviewed papers that were published during my time as a technician in Claudia Wellbrock’s research group. Between them they explore the cellular response to targeted therapies in melanoma. The key findings from each paper and how these contributed to the wider field of research will be discussed. The first paper characterised the effects of the MEK inhibitor (MEKi) AZD6244 on melanoma cell growth and invasion. It was found that although the MEKi inhibited proliferation and induced cell death, it also unexpectedly promoted integrin mediated invasion. Treatment with the SRC inhibitor AZD0530 was able to block this increase by de- stabilising the integrin complexes integral for invasion. Combinatorial MEK and SRC inhibition was therefore proposed as a new approach to the treatment of melanoma. The second paper explored the limitations of MEKi mono-therapy such as dose limiting toxicity and drug resistance. We identified the melanoma master regulator MITF as the crucial factor providing survival signals in the presence of MEKi and hence contributing to drug resistance. TGF-β signalling was found to sensitise cells to MEKi through a reduction in the expression of MITF and its up-stream regulator PAX3. As direct TGF-β treatment has been shown to promote invasion, the pathway was indirectly targeted through inhibition of the E3 ubiquitin ligase SMURF2. This effectively reduced MITF expression and sensitised cells to MEK inhibition both in vitro and in vivo. In the third paper, we explored the effect of the immune microenvironment on MAPK pathway inhibition. Treatment with MAPKi promoted macrophage infiltration at the tumour site, driving an increase in stromal derived TNFα. This resulted in an up-regulation of MITF, promoting melanoma cell growth, survival and therapy resistance. IkB-kinase (IKK) inhibitors effectively disrupted TNFα induced MITF expression and when used in combination with MEKi, enhanced cell death both in vitro and in vivo. The fourth paper investigated drug tolerance in melanoma cells; a state characterised by the MAPKi induced up-regulation of PAX3 and MITF expression. An FDA approved drug screen identified nelfinavir as a compound able to reduce the levels of both MITF and PAX3 and sensitise cells to MAPKi both in vitro and in vivo. The fifth paper examined tumour heterogeneity and its impact on response to MAPKi therapy. Intra-tumour heterogeneity of MITF expression is maintained throughout BRAF inhibitor (BRAFi) treatment. We demonstrated that BRAFi induced MITF up-regulation in the MITF-high phenotype is able to confer paracrine resistance via EDN1 secretion. Disruption of this signalling using various EDN1 receptor antagonists sensitised tumours to BRAF inhibition. This approach was found to target the two major phenotypes (MITF- high/AXL-low and MITF-low/AXL-high), making it an attractive approach to increase the longevity of BRAF inhibitor response in patients. The final paper considered how the metabolic state of melanoma cells impacts on MITF mediated proliferation. We demonstrated that melanoma cells are dependent on glucose for proliferation and that this response is regulated through the transcription factor MITF. Luciferase reporter assays revealed that CREB and ATF4 are competing transcription factors that regulate MITF transcription in response to changes in glucose availability.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Claudia Wellbrock (Supervisor)