Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Natalie G. Ahn

Second Advisor

William M. Old

Third Advisor

Tin Tin Su

Fourth Advisor

Xuedong Liu

Fifth Advisor

Dylan J. Taatjes

Abstract

Skin cancer is the most commonly diagnosed cancer in the U.S. and 75% of skin cancer related deaths are due to malignant melanoma, a cancer originating in melanin producing melanocytes. The RAF/MKK/ERK signaling cascade is constitutively activated in over 90% of melanomas and 52% of tumors contain BRAF V600E/K oncogenic driver mutations. Although small molecule inhibitors specifically targeting mutant BRAF V600E/K and the downstream kinases MKK1/2 have been successful in clinical settings, resistance invariably develops. In preclinical studies, inhibitors of ERK1/2 can overcome resistance to BRAF V600E/K and MKK1/2 inhibitors, making them promising alternative pathway inhibitors for the treatment of melanoma. However, the specificity of molecular responses to ERK1/2 inhibitors remains unknown. In this thesis, I use SILAC-based phosphoproteomics to quantify molecular responses to the clinically available MKK1/2 inhibitor, trametinib, and the ERK1/2 inhibitors, SCH772984, GDC0994 and Vertex-11e in WM239a human metastatic melanoma cells. I observed significant responses in approximately 5% of all phosphosites identified. Significantly regulated phosphosites showed a high degree of overlap between all inhibitors, suggesting that the pathway functions linearly with relatively little evidence for branchpoints that lead to bifurcation upstream of ERK1/2. I also observe phosphosites responsive to only one of four MKK1/2 or ERK1/2 inhibitors. For example, trametinib shows an ability to block activating phosphorylation sites on p38α MAPK, which are not shared by ERK1/2 inhibitors SCH772984 and GDC0994, the MKK1/2 inhibitor selumetinib, or the BRAF V600E/K inhibitor, vemurafenib. Trametinib directly inhibits MKK6 in vitro, although with a IC50 10-fold higher than its inhibition of p38α MAPK in cells. This suggests the potential that the direct target of trametinib in cells is upstream of MKK6. Further analyses of phosphoproteomics responses to two MKK1/2 inhibitors and two ERK1/2 inhibitors identifies phosphorylation sites that can be classified as (i) known or novel targets of BRAF-MKK-ERK signaling, (ii) potential branchpoints at MKK1/2 upstream of ERK1/2, and (iii) off-targets of different inhibitors. My results show how information from phosphoproteomics comparisons of multiple MKK1/2 and ERK1/2 inhibitors can be combined to provide a deeper understanding of pathway specificity.

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