Sir Peter MacCallum Department of Oncology - Theses
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ItemInvestigating therapeutic strategies targeting metabolism in NRAS-mutant melanoma( 2017)In human cancers, RAS mutations are among the most commonly identified mutations; however therapies targeting RAS remain elusive. Recent investigations have demonstrated that mutated RAS can reprogram metabolism in cancer cells. In the field of melanoma, the role of mutant BRAF in the regulation of metabolism has also been elucidated. However, surprisingly little is known about the importance of mutant NRAS in melanoma metabolism. Alongside this, there are limited targeted therapies available for the treatment of patients with NRAS-mutant melanoma. Consequently, this thesis aims to characterise the metabolic response of RAS-mutant cells to targeted therapies and to use this knowledge to develop novel therapeutic strategies targeting glucose metabolism in NRAS-mutant melanoma. Using human cancer cell lines, the studies in this thesis demonstrate that a number of similarities exist between NRAS and BRAF-mutant melanoma cells with respect to their metabolic responses to MAPK pathway inhibition. MEK inhibition, consistent with prior reports of the effects of BRAF inhibition in BRAF-mutant melanoma, resulted in suppression of glycolysis evidenced by decreased lactate production, glucose uptake and extra-cellular acidification rate. Importantly, known transcriptional regulators of glycolysis in BRAF-mutant melanoma (HIF1α, MYC and MondoA) also play a role in the response of NRAS-mutant melanoma cells to MEK inhibition. Furthermore, in the setting of MEK inhibition NRAS-mutant melanoma cells have increased oxidative metabolism, with increased PGC1α and MITF expression. This adaptation has previously been reported in BRAF-mutant melanoma. The studies in this thesis investigated the relative importance of the MAPK and PI3K effector pathways of RAS, demonstrating that MAPK pathway inhibition had the most consistent and significant effects on glucose metabolism in NRAS-mutant melanoma cells. Finally, a comparison of NRAS and KRAS-mutant cells revealed that NRAS-mutant cells are more sensitive to MEK inhibition, with a more pronounced reduction in parameters relating to glycolysis. Given these findings, it was hypothesised that combining a MEK inhibitor with an inhibitor of glucose metabolism would be an effective therapeutic strategy in NRAS-mutant melanoma. To this end, the MEK inhibitor trametinib was combined with the mitochondrial inhibitor PENAO. In vitro, PENAO enhanced the anti-proliferative activity of trametinib in NRAS-mutant melanoma cells, with additive effects on glycolysis and mitochondrial metabolism. In vivo, the combination was well tolerated, however the addition of PENAO did not enhance the effect of trametinib on tumour growth. These studies are important in demonstrating the feasibility of a combination targeting two key metabolic processes in vivo, particularly when one process is under the control of an oncogenic aberration. A chemical screen to identify combinations that enhance the suppression of glycolysis achieved by MEK inhibition has been commenced. In summary, this work has characterised important metabolic adaptations in the context of MEK inhibition in NRAS-mutant melanoma. Although an effective targeted therapy for RAS remains elusive, this research supports the ongoing exploration of strategies that target RAS effector pathways in combination with key metabolic processes, particular in the context of NRAS-mutant melanoma.
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ItemEnhancing adoptive immunotherapy: redirecting immune subsets and metabolic pathways( 2017)The adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) as a treatment for cancer has achieved impressive responses in haematological malignancies, but has been less successful in the treatment of solid tumors. The tumor microenvironment of solid tumors presents multiple forms of immunosuppression, inhibiting the efficient effector function of infiltrating anti-tumor T cells. During my PhD, we assessed the potential of two strategies to enhance the anti-tumor function of CAR T cells. The first focuses on the potential of other CAR-expressing immune subsets to stimulate CAR T cell function and persistence in the tumor microenvironment. To elucidate the function of CAR-expressing non-T lymphocytes, we generated a transgenic mouse model (vav-CAR) in which immune cells express a CAR against the Her2 (ErbB2) tumor antigen. As expected, CAR T cells harboured anti-tumor function but we also found that CAR-modified macrophages and natural killer cells (NKs) exhibited significant antigen specific cytokine secretion, cytotoxicity and phagocytosis. Moreover, using the vav-CAR model, we demonstrated the potential of CAR immune cells to mediate tumor rejection independently of CD8+ T cells. CD4+ T cells were critical for this response as their deletion severely abrogated the anti-tumor responses in our vav-CAR model. Distinct T helper subsets have been shown to participate to anti-tumor responses, with Th1 and Th17 cells demonstrating a more robust efficacy as compared to other T helper subsets. Our second strategy was focused on the impact of metabolism in the polarisation of CD4+ T cells, in particular the differentiation of CAR T cells to Th1 lineage. T cell activation and polarisation is highly associated with increased metabolic needs. Given that nutrient deprivation in the tumor microenvironment, due to a high demand of the tumor for resources, can limit the nutrients available for other cell types, the fate and function of adoptively transferred immune cells may be altered upon entering the tumor. Therefore, modifying CAR immune cells to resist metabolic suppression in the tumor microenvironment may help retain their effector functions. Upon assessing the effects of nutrient deprivation on T cell differentiation, we previously found that limiting concentrations of glutamine, the most abundant amino acid in the plasma, inhibited the potential of T cells to undergo Th1 differentiation with associated IFNγ secretion. Rather, this condition resulted in the conversion of naïve CD4+ T cells into suppressive FOXP3+ regulatory T cells (Tregs). Here, we determined that a single glutamine-derived metabolite, α-ketoglutarate (αKG), enhanced the anti-tumor effector functions of multiple CAR T helper subsets, increasing the production of IFNγ and reducing FOXP3 expression. Thus, during my PhD, I generated a vav-CAR model, providing a platform in which the function of multiple CAR-bearing immune subsets can be studied and manipulated. This model will promote the utilisation of optimized CAR-bearing immune cells in adoptive immunotherapy for solid tumors. Furthermore, using the CAR model, we have identified a glutamine metabolite that orchestrates immune responses through the metabolic reprogramming of CD4+ T cells.