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Identifying and characterising transmembrane transporters of tryptophan and kynurenine in normal and cancerous brain tissue

Final Report Summary - KP TRANSPORT (Identifying and characterising transmembrane transporters of tryptophan and kynurenine in normal and cancerous brain tissue)

The body’s immune system holds the incredible potential to specifically destroy a tumour without causing damage to normal tissue, while also providing long-term resistance to tumour recurrence through immunological memory. In recent years research in immuno-oncology has shown that cancers employ several mechanisms to evade the body’s immune system. Cancer cells can become ‘invisible’ to the immune system by not expressing the key ‘HLA’ proteins which are required for mediating interactions between immune cells and normal cells, they can ‘exhaust’ immune cells by falsely signalling that an immune response is no longer required, and they can generate a local microenvironment around the cancer tissue which prevents immune cells from functioning.

This project initially proposed to investigate how a subset of breast and brain tumours modify their microenvironment to suppress the immune system using kynurenine. Tumours expressing high levels of the enzymes TDO2, IDO1 or IDO2 rapidly metabolise the essential amino acid tryptophan into kynurenine, thereby depriving nearby immune cells of the tryptophan they require for their normal function. Within the tumour cells kynurenine triggers a signalling cascade that results in the activation of DRE response genes, making tumour cells more aggressive and likely to metastasise. Furthermore, tumours export kynurenine into their microenvironment where it supresses the function of local immune cells by co-opting the body’s natural mechanism for signalling that an immune response is no longer required.

The transporter proteins responsible for the export of kynurenine from tumour cells are not fully understood, yet represent viable therapeutic targets not just for cancer, but other disorders in which the levels of tryptophan-derived metabolites are misregulated, notably in autoimmune disorders such as multiple sclerosis.

The first hurdle for this project was therefore the accurate measurement of tryptophan and kynurenine; while it is possible to measure the concentration in samples using high performance liquid chromatography (HPLC), this cannot be done in live cells, and we found that the existing tryptophan nanosensor we had envisaged for use in this project simply was not robust enough for measurements in cells or indeed live animals. A significant challenge for this project has been the development of molecular reporter constructs that can be used to indicate local concentrations of Trptophan. We have been able to create reporter that measured tryptophan directly by repurposing bacterial tryptophan binding proteins, as well as indirectly by measuring the transcription of genes under control of the kynurenine sensitive transcription factors AhR/ARNT. These reporters have been used in numerous collaborative projects in the lab, including with a pharmaceutical partner, and will represent the bulk of the deliverables relating to the initial aims. Due to the significant time required to develop these tools, which had initially been thought to be in place, relatively limited progress has been made on the screening of transporters, although this is now ongoing in a high throughput fashion and will continue beyond the end of the reporting period.

The original proposal also included a number of ‘high risk/high reward’ projects. The first such project set out to explore the potential of ‘humanized’ Drosophila as a drug dosage testbed. As described in the mid term report, some of this work had already been performed by another group within the DKFZ with negative results. Furthermore, it is clear that this type of platform is no longer valued by industry, therefore limited efforts were made to further develop this project. The second such project involved the transduction mammalian cells with the Drosophila gene cardinal, a phenoxazinone synthetase that converts the breakdown product of 3-hydroxy-kynurenine (itself derived from kynurenine) into a coloured ommochrome susbtrate. Such a cell line would also be an appropriate drug testing platform, however despite considerable efforts we were unable to obtain pigment in cells, potentially due to mammalian cells missing important cofactors for cardinal protein.

Since this project was conceived the field of cancer immunotherapy has shifted focus from the microenvironment, and put greater emphasis on so/called ‘checkpoint inhibitor’ monoclonal antibody therapies that prevent tumour cells from exhausting immune cells. However, tumours with fewer mutations (such as brain tumours) do not respond to such therapy without the immune system first being primed. This priming process is almost entirely unique not only to every patient, but also to every tumour within that patient: a personalised immunotherapy.

This technology is at the forefront of cancer research, however due to the significant financial outlay per patient and limiting technical expertise such therapies are only researched in a very small number of cancer institutes around the world, including the DKFZ. Over the last 6 months of this fellowship we have leveraged the DKFZ’s investment in sequencing and become deeply involved in this rapidly evolving field, which complements this project’s research on targeting the cancer microenvironment. The fellow has contributed significant computing expertise in the lab, and played a key role in helping to implement a pipeline to design patient-specific cancer vaccines, and while much of this work is establishing the potential of this therapy in a research setting, the vaccine designs resulting from our work – after a short period of testing in the lab – are being offered to brain tumour patients, making a direct impact on the lives of people for whom current cancer therapy offers little hope.

The fellow has also engaged fully with the DKFZ postdoctoral training scheme, taking numerous courses both scientific (e.g. FELASA animal testing certification) and training for his future career as a group leader (e.g. leadership training), and begun to give invitational talks at international meetings, further expanding his scientific network.