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An integrative approach for the exploration of melanoma genetic and immunological interactions

Periodic Reporting for period 2 - MEL-Interactions (An integrative approach for the exploration of melanoma genetic and immunological interactions)

Reporting period: 2020-04-01 to 2021-09-30

Melanomas until recently had limited therapeutic options. Immunotherapy, has gained prominence as a therapeutic modality. Despite the high response rate to immunotherapy, it is necessary to decipher biomarkers that predict response to immunotherapy and assess resistance mechanisms towards immunotherapy. To this end, we established a mouse melanoma model to investigate whether tumor mutational burden as well as intratumor heterogeneity affect aggressive tumor cell growth as well as response to immune checkpoint blockade. We identified the antigens that are recognized by tumor infiltrating T cells using HLA peptidomics. This allowed us to identify novel neoantigens, bacteria-derived antigens as well as aberrant peptides derived from translation mistakes, categories of antigens which are further described below.
We focused on the interferon-gamma (IFN‐γ), a cytokine that induces the activity IDO1, which stimulates the production of tryptophan. Along with another amino acid called kynurenine, tryptophan influences the immune response. We know that depletion of tryptophan levels, alongside a parallel accumulation of kynurenine, leads to immunosuppression, but the mechanism leading to the immunosuppression is not fully understood. We used ribosome profiling in melanoma to investigate the effects of IFN‐γ treatment on mRNA translation. We discovered that the depleted levels of tryptophan associated with immunosuppression is associated with frameshifting which occurs during translation that results in the production of multiple unique proteins from a single mRNA. We demonstrated that, after treatment with IFN‐γ, frameshifting led to the generation of aberrant peptides at the cell surface. Our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFN-γ, has a role in the immune recognition of melanoma cells by contributing to diversification of peptides that can be presented and targeted by T cells. We further reported on how bacteria that reside within tumor cells can be harnessed to provoke an immune reaction targeting the tumor. This discovery offers a new avenue for improved cancer immunotherapy and explains the findings of previous research showing that the gut microbiome affects immunotherapy success.
The combination of these discoveries have allowed us to better define the HLA-peptidomic landscape and provide an avenue for personalized treatment using cancer vaccination.
NRAS mutant patients have a low survival rate. MAPK pathway targeted therapies, such MEK inhibitors are currently used to treat NRAS mutant patients. However, the response to these inhibitors is variable due to resistance. We employed a CRISPR-cas9 knockout screen that lead to the identification of the novel target FBXO42 involved in NRAS mutant melanoma-acquired resistance to the MEK1/2 inhibitor trametinib. We found that combining trametinib with takinib had a synergistic effect. These data provide new insights into RAS pathway regulation and provides opportunities for novel therapeutics.
We also assessed heterogeneity and neo-antigen/mutational load as independent factors affecting immune response and tumor growth in animal models. Our main findings were that (i) A UVB-induced increase in tumor heterogeneity gives rise to increased tumor growth in vivo accompanied by a dampened immune response, despite an increase in mutational load. In contrast, highly homogenic single-cell clones derived from the same UVB-exposed cell line are highly immunogenic and undergo immune rejection, regardless of their mutational load. (ii) these results were in concordance with those we observed while assessing melanoma patient data. Our results suggest that assessing tumor heterogeneity should play a larger role in stratifying patients to checkpoint immunotherapies.
Another one of our projects dealt with bacteria which have been demonstrated to colonize human tumors. Our microbial analysis of melanoma patient metastases revealed that intra-tumor bacteria are able to enter patient-derived melanoma cells, be presented by tumor cells and elicit immune-reactivity, not only yielding a comprehensive analysis of melanoma intra-tumor bacteria and HLA-presentation co-signatures, but also providing insights on how bacteria influence the activation of the immune system and response to checkpoint inhibitors.
T-cell mediated immunotherapy is commonly based on T-cell receptor-specific recognition of peptides presented on MHC molecules. Efforts seeking to discover tumor antigens mostly focus on germline or differentiation antigens and peptides coded by mutated genes. However, accumulating evidence show that endogenous peptides originating from non-canonical mRNA translation also play a role in protective immunity. Importantly, when cells adapt to nutritional constrains and stress conditions, dramatic changes in the translatome occur, which may be via translational aberrations. We have recently demonstrated that environmental cues reprogramming the translation machinery drive the generation and MHC-presentation of aberrant peptides. We have also uncovered that melanoma cells may present peptides derived from intra-tumor bacteria which also affect the patient immune response. To identify these new categories of presented antigens, we needed to generate novel experimental tools to understand how non-genomic-based attributes integrate with the peptidome landscape. We further used novel tools to unvail the interplay between cancer cells and the immune system. For this we used state of the art analysis of the human proteome, HLA-presentation and cognate T-cell identification as well as in vivo models for investigation of the immune system.
Additional peptides, derived from adaptive, non-canonical translation may be involved in immunosurveillance; which are yet to be identified. We aim to continue our investigation of the cancer immunopeptidome using additional state of the art tools, such as analyses of nascent peptide production and evaluation of tRNA populations at particular stress conditions and integrating these data with novel computational methods.
imaging of melanoma cells that were infected with bacteria
immunofluorescence images of tumor blocks with bacteria