Investigating the metabolic and therapeutic effects of combined glutaminase and mutant-BRAF inhibitors in melanoma

Harris, Rachel (2022) Investigating the metabolic and therapeutic effects of combined glutaminase and mutant-BRAF inhibitors in melanoma. PhD thesis, University of Glasgow.

Abstract

Metastatic melanoma is a deadly form of skin cancer and the leading cause of skin cancer deaths. For patients with stage three and four melanomas, an adjuvant therapy known as Vemurafenib is used as a first line therapy for all patients whose tumour harbours a BRAF mutation. This treatment is an inhibitor of the mutant BRAF oncoprotein and prevents the constitutive activation of the RAS-RAFMEK-ERK signalling cascade. This treatment allows remarkable clinical success, but BRAF inhibitor resistance invariably develops, however previous studies showed that acquisition of BRAF inhibitor resistance causes increased dependence on glutamine.

Therefore, in this study I investigated the use of the clinically relevant glutaminase inhibitor, CB839 in mutant BRAF melanoma. I demonstrated that glutaminase inhibition selectively reduces growth of cells in vitro and tumours in vivo under long term BRAF inhibition. However, I found that the presence of BRAF inhibition itself sensitizes some of these models to glutaminase inhibition. BRAF inhibitor resistant cells under BRAF inhibition in vitro had reduced generation of glutamine derived glutamate. In addition, BRAF inhibition in vivo resulted in decreased transcription of genes encoding for glycolytic enzymes and transporters. These transcriptional changes were associated with decreased pyruvate and ATP/AMP ratio when BRAF inhibition was coupled with glutaminase inhibition.

In vitro, glutaminase inhibition caused decreased the production of glutamine derived aspartate, which was shown to be essential for the growth of these cells. In vivo, tumour aspartate levels were decreased by glutaminase inhibition, but unexpectedly increased by BRAF inhibition, demonstrating that aspartate levels obtained under CB839 treatment do not limit the growth of melanoma xenografts.

Further investigation in vivo revealed that BRAF inhibition causes metabolic rewiring, increasing the tumour levels of glutamine and decreasing those of citrulline. Furthermore, BRAF inhibition increased the tumour expression of argininosuccinate lyase (ASL), and decreased the levels of systemic ammonia, suggesting that BRAF inhibition may affect the activity of the urea cycle. In vivo, an untargeted metabolomic approach found several metabolic features whose levels were altered by combined CB839 and PLX4032 treatment. Further studies to confirm their identities and their role in the antitumour effect of CB839 and PLX4032 treatment may yield useful insights into the use of the combination of these two clinically relevant treatments.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Supervisor's Name: Sumpton, Dr. David and Tardito, Dr. Saverio
• Date of Award: 2022
• Depositing User: Theses Team
• Unique ID: glathesis:2022-83354
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Jan 2023 14:23
• Last Modified: 12 Jan 2023 14:23
• Thesis DOI: 10.5525/gla.thesis.83354
• URI: https://theses.gla.ac.uk/id/eprint/83354