Approximately 80,000 Americans are diagnosed with melanoma each year, leading to over 9,000 deaths. Of these, approximately 80% harbor activating mutations in the MAPK pathway signaling protein BRAF;most commonly a V600E substitution. In 2011, the FDA approved the drug vemurafenib, a selective BRAF Ser/Thr kinase inhibitor, based on phase III trials showing a median progression-free survival benefit of 5.3 months in patients with late-stage BRAF-mutated melanoma. FDG-PET, which uses uptake of a radioactive glucose analog as a proxy for metabolic activity, is used to image melanoma in the clinic due to the tumors'highly elevated glucose metabolism relative to normal tissue. Though the majority of patients show a dramatic decrease in PET positivity within two weeks of beginning therapy, in 40% of these patients, less than 30% maximal tumor shrinkage is observed in spite of the survival benefit. Furthermore, in the majority of these patients, tumor recurrence occurs in between 2 and 18 months, with a corresponding increase in PET positivity, signifying tumor resistance to the drug. Our preliminary data suggest that melanoma cells grown in the presence of vemurafenib show a decrease in glucose uptake and proliferation, associated with a significant decrease in hexokinase activity. Based on these observations, we hypothesize that the effects of vemurafenib on PET positivity and progression-free survival are partly due to vemurafenib-induced decreases in glucose metabolism.
Specific aims : This proposal is comprised of two specific aims.
The first aim i s focused on quantifying the vemurafenib-induced decrease in glucose uptake in a large number of BRAF-mutant sensitive and resistant melanoma cell lines. We will use fluorescent glucose analog-based flow cytometry, as well as analysis of changes in transmembrane glucose transport using a 3H-O-methyl-D-glucose uptake assay, changes in hexokinase activity and localization, and RNAi to functionally evaluate the importance of individual hexokinase and glucose transporter genes. We will also evaluate glutamine as an alternative metabolic substrate through isotopic labeling and flux analysis, as well as glucose and glutamine deprivation experiments.
The second aim will focus on further exploring preliminary data identifying altered hexokinase biology as a possible mechanism for the observed decrease in glucose uptake. We will use immunoprecipitation, mass spectrometry, Western blotting, RNAi, and confocal microscopy to evaluate changes in the MAPK, AKT, and GSK3? signaling pathways, changes in post-translational modification, and altered binding of isoforms to VDAC mitochondrial membrane channels, as well as other proteins, as potential causes of decreased activity. Relevance: This project will not only evaluate the currently-unexplored metabolic effects of vemurafenib, but may also yield valuable information on the translational correlates of FDG-PET imaging in the setting of BRAF inhibition. New discoveries in the mechanisms underpinning metabolic changes may also suggest new metabolically-based combination therapies aimed at overcoming vemurafenib resistance.
Melanoma is the most lethal form of skin cancer. BRAF kinase inhibitors that produce high rates of clinical response in melanoma, but are also associated with development of resistance in most cases. Response to BRAF inhibition in melanoma is characterized by markedly reduced glucose uptake, a characteristic that is lost upon emergence of resistance. In this proposal we examine the mechanisms of BRAF modulation of glucose uptake to identify new approaches to treating resistant melanoma.