Periodic Reporting for period 4 - Breakborder (Breaking borders, Functional genetic screens of structural regulatory DNA elements)
Reporting period: 2024-03-01 to 2024-08-31
Our major discovery in this Breakborder project was the identification of a novel phenomenon in cancer biology. While investigating the relationship between cancer cells and the immune system, we noticed that cancer cells could continue producing proteins, albeit with a tryptophan shortage. When T cells recognize cancer cells, they secrete interferon-gamma (IFNg) that triggers the induction of IDO1, an enzyme that degrades tryptophan, an essential amino acid. We showed that cancer cells are “sloppy” – they continue to produce proteins under tryptophan shortage, resulting in aberrant protein production. These aberrant proteins are processed into peptides that are presented at the surface of the cancer cells to the immune cells. We also demonstrate that these aberrant peptides are targetable and can be used to strengthen immunotherapeutic approaches. We conclude that our findings have not only pinpointed an exciting new biology never described in human cells before but also have a great potential societal impact on improving immunotherapy for cancers that have become resistant to such treatment.
Using CRISPR technology, we first targeted regulatory regions in the genome linked to estrogen receptor-driven breast cancer cells. Our data, published in NAR Oct 2019, showed that such regions are required for hormone-driven breast cancer cell proliferation.
Second, we performed a comprehensive CRISPR screen for regulatory regions in our genome that are targets of the important oncogene. In a publication in Genome Biology Journal 2021, we elucidated how YAP induces cancer aggressiveness (progression and metastasis) through specific regions and chromatin structure, assisting in diagnosing cancer metastasis.
Third, the results obtained from this project and the tools made here were used to connect gene expression to the emergence of new mRNA isoforms. We uncovered a novel form of mRNA with translation potential and showed that these novel mRNA isoforms are important for cell survival and communication with the immune system.
Finally, this resource was used to initiate a new and completely novel research line examining the quality of proteins produced in cancer cells under nutrient shortage induced by immune cell attack. In a set of 3 published papers (2x Nature and 1x Molecular Cell, 1x Science, and 2x review papers in Molecular Cell and Oncogene) we uncovered the process of aberrant production of proteins in cancer cells in times of nutrient shortage. This novel phenomenon enriches the neo-epitope landscape of cancer cells and can help improve cancer immunotherapy. This line of research is absolutely novel, extremely interesting from a biological perspective, and important for advancing cancer immunotherapy further.
One aspect of exploitation of our finding is the identification of T cell receptors (TCRs) that target aberrant neoepitopes in a cancer-specific manner. We already have an in-press follow-up manuscript demonstrating that such an adoptive T-cell therapy approach might be feasible. A second exploitation aspect of this work is investigating whether and how such mechanisms are regulated at the molecular level. We already have secured an ERC advanced grant to follow this route in the next 5 years. Mainly, we would like to know if other types of shortages induce aberrant protein production in cancer and how aberrant protein production is regulated. Can aberrant protein production assist cancer cell survival? And can we find ways to modulate it to kill cancer cells?
Second, we performed a comprehensive CRISPR screen for regulatory regions in our genome that are targets of the important oncogene. In a publication in Genome Biology Journal 2021, we elucidated how YAP induces cancer aggressiveness (progression and metastasis) through specific regions and chromatin structure, assisting in diagnosing cancer metastasis.
Third, the results obtained from this project and the tools made here were used to connect gene expression to the emergence of new mRNA isoforms. We uncovered a novel form of mRNA with translation potential and showed that these novel mRNA isoforms are important for cell survival and communication with the immune system.
Finally, this resource was used to initiate a new and completely novel research line examining the quality of proteins produced in cancer cells under nutrient shortage induced by immune cell attack. In a set of 3 published papers (2x Nature and 1x Molecular Cell, 1x Science, and 2x review papers in Molecular Cell and Oncogene) we uncovered the process of aberrant production of proteins in cancer cells in times of nutrient shortage. This novel phenomenon enriches the neo-epitope landscape of cancer cells and can help improve cancer immunotherapy. This line of research is absolutely novel, extremely interesting from a biological perspective, and important for advancing cancer immunotherapy further.
One aspect of exploitation of our finding is the identification of T cell receptors (TCRs) that target aberrant neoepitopes in a cancer-specific manner. We already have an in-press follow-up manuscript demonstrating that such an adoptive T-cell therapy approach might be feasible. A second exploitation aspect of this work is investigating whether and how such mechanisms are regulated at the molecular level. We already have secured an ERC advanced grant to follow this route in the next 5 years. Mainly, we would like to know if other types of shortages induce aberrant protein production in cancer and how aberrant protein production is regulated. Can aberrant protein production assist cancer cell survival? And can we find ways to modulate it to kill cancer cells?
Our prime discovery was the realization that cancer cells produce aberrant proteins and present them as neo-epitopes to the immune system. While further investigation into the regulatory mechanisms that govern aberrant protein production and the exploitation of their resulting neo-epitopes is currently ongoing, our discoveries show progress beyond state-of-the-art and connect basic events in the field of mRNA translation to cancer immunology in a manner never anticipated before.
We showed that T cell-induced tryptophan shortage stimulates cancer cells to produce out-of-frame mRNA translation due to ribosomal collisions at tryptophan codons as well as codon reassignments (phenylalanine to tryptophan W>F). The resulting aberrant proteins are processed and presented to the immune system as neoepitopes. Further, we demonstrated that these events are enriched in the proteomes of cancer cells compared with adjacent normal tissues and that oncogenic mutations in the MAP kinase pathway drive them. Further, we show that arginine deprivation in lung cancer enriches their proteomes with cysteine by a tRNA misalignment mechanism. This event is linked to oncogenic mutations in the ferroptosis pathway and enhances the survival of cancer cells due to the high capacity of cysteine to scavenge toxic oxygen radicals. Finally, we showed that chemotherapy triggers p53-independent apoptosis by inducing tRNA-leucine shortage. DNA damage induces the activity of SLFN11 protein that cleaves tRNA-UAA and stalls ribosomes at TTA-leucine codons, triggering extensive ribosome collisions and cell death. This is important. In a new manuscript (in press), we show that TTA-leucine stalling by chemotherapy induces aberrant protein production, resulting in neo-epitopes presentation that can be recognized by T cells to promote cancer cell killing.
We showed that T cell-induced tryptophan shortage stimulates cancer cells to produce out-of-frame mRNA translation due to ribosomal collisions at tryptophan codons as well as codon reassignments (phenylalanine to tryptophan W>F). The resulting aberrant proteins are processed and presented to the immune system as neoepitopes. Further, we demonstrated that these events are enriched in the proteomes of cancer cells compared with adjacent normal tissues and that oncogenic mutations in the MAP kinase pathway drive them. Further, we show that arginine deprivation in lung cancer enriches their proteomes with cysteine by a tRNA misalignment mechanism. This event is linked to oncogenic mutations in the ferroptosis pathway and enhances the survival of cancer cells due to the high capacity of cysteine to scavenge toxic oxygen radicals. Finally, we showed that chemotherapy triggers p53-independent apoptosis by inducing tRNA-leucine shortage. DNA damage induces the activity of SLFN11 protein that cleaves tRNA-UAA and stalls ribosomes at TTA-leucine codons, triggering extensive ribosome collisions and cell death. This is important. In a new manuscript (in press), we show that TTA-leucine stalling by chemotherapy induces aberrant protein production, resulting in neo-epitopes presentation that can be recognized by T cells to promote cancer cell killing.