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As cancers develop and spread they must continually evade immune destruction. Understanding mechanisms of immune evasion in cancer is clinically significant as demonstrated with the successes of immune checkpoint inhibitors. Breast cancer is known to be highly immune evasive and responds poorly to the current immunotherapies, indicating alternative immune pathways must be targeted. We hypothesise that there are unidentified genetic mechanisms that enable immune evasion in breast cancer. We aim to uncover and target these mechanisms to sensitise immune evasive breast cancer cells to immune destruction in the context of immunotherapy treatment. DNA barcoding technology offers a new approach to understanding immune evasion. By stably integrating a unique DNA barcode sequence into each cell, we can study clonal immune evasion in vivo. Using this technology, we identified cancer cell clones from the 4T1 murine mammary carcinoma cell line that are highly enriched in lung metastases following treatment with combination immunotherapy (anti-CTLA-4 plus anti-PD-1). We isolated these specific immune evasive clones and established them as clonal cell lines. We have identified stark clonal differences in both PD-L1 and MHC I expression at both the RNA and protein level, and shown that MHC I expression is only partially controlled by epigenetic mechanisms. In addition, immune evasive subclones co-cultured with stimulated T cells resulted in less activated T cells than their less evasive counterparts. Furthermore, RNA sequencing of these clones has identified a gene signature that is strongly associated with decreased survival in both the METABRIC and TCGA cohorts. We have demonstrated ongoing immunoediting in the 4T1 model in vivo, both during metastasis and immunotherapy treatment. We have also identified subclonal populations of cells within a single tumour utilising different mechanisms of immune evasion. RNA sequencing has revealed a gene signature strongly associated with poor survival of basal-like breast cancer in two cohorts. Further pathway-level analysis of the resulting gene signature is required to elucidate the drivers of this aggressive and immune evasive phenotype. By targeting newly identified mechanisms of immune evasion in combination with current immunotherapies, we hope to improve the long-term survival of breast cancer patients. Citation Format: Louise A Baldwin, Nenad Bartonicek, Jessica Yang, Sunny Z Wu, Niantao Deng, Daniel Roden, Chia-Ling Chan, Ghamdan Al-Eryani, Damien J Zanker, Belinda S Parker, Alexander Swarbrick, Simon Junankar. Dna barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-04.