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Background Breast cancer is the leading cause of cancer mortality among females. 1 APOBEC3 (A3) enzymes, specifically A3A and A3B, deaminate cytosine to uracil in single-stranded DNA, leading to diagnostic mutational signatures in 15-20% of primary tumors and 25-30% of metastases.2 A3 expression and mutagenesis associate with poor clinical outcomes including therapy resistance.2 However, a potential relationship between A3 mutagenesis and immunotherapy treatment responsiveness has yet to be investigated in murine models.Methods Two mammary cancer cell lines, MM001i and MM008i, were isolated from spontaneously arising tumors in C57BL/6J animals with mammary specific heterozygosity for p53. Tumors were orthotopically implanted in wildtype (WT) C57BL/6J mice, followed by treatment with αPD-1 and/or αCTLA-4. Tumor response, rechallenge immunity, immune cell infiltration, and transcriptomic signatures were evaluated by caliper measurements, flow cytometry, IHC, bulk RNA-seq, and single-cell RNA-seq (scRNA-seq). Cells were engineered to express human A3A or its catalytic mutant E72A, followed by single clone isolation and analysis of mutational signature, predicted neoantigen load, and immunotherapy response.Results MM001i and MM008i both engraft robustly in the mammary fat pad of WT mice. MM008i tumors demonstrated complete regression across all immunotherapy treatment groups, whereas MM001i tumors responded only to αCTLA-4. Both tumor cell lines also triggered robust memory responses. Flow cytometry and IHC revealed clear differences in immune infiltration: MM008i tumors exhibited dense CD3 + T cell infiltration (particularly activated, pre-exhausted PD-1+Tim3-CD8+ T cells), and MM001i tumors were comparatively immune-excluded. Comparative transcriptomic analysis showed enrichment in extracellular matrix remodeling and inflammatory pathways. A3A clones were significantly enriched with A3A mutational signatures SBS2 and SBS13 versus E72A clones. Mutated clones were still able to successfully engraft in WT mice. Neoantigens were identified by computational prediction.Conclusions MM001i and MM008i are novel syngeneic mammary cancer models with similar responses to αCTLA-4 and divergent responses to αPD-1. MM008i had significant tumor infiltrating lymphocytes unlike MM001i. An A3A mutagenized derivative with an expanded neoantigen repertoire still engrafts into WT animals, which enables additional studies on differential immunotherapy responses. Together, these new syngeneic models provide tools to improve our understanding of the immune microenvironment and test therapeutic strategies for A3 hypermutated tumors.References Azamjah N, Soltan-Zadeh Y, Zayeri F. Global trend of breast cancer mortality rate: A 25-year study. Asian Pac J Cancer Prev. 2019;20:2015–2020.Gupta A, Gazzo A, Selenica P, Safonov A, Pareja F, da Silva EM, Brown DN, Shao H, Zhu Y, Patel J, Blanco-Heredia J, Stefanovska B, Carpenter MA, Chen Y, Vegas I, Pei X, Frosina D, Jungbluth AA, Ladanyi M, Curigliano G, Weigelt B, Riaz N, Powell SN, Razavi P, Harris RS, Reis-Filho JS, Marra A, Chandarlapaty S. Apobec3 mutagenesis drives therapy resistance in breast cancer. Nat Genet. 2025;57:1452–1462.Ethics Approval All animal studies were approved by The University of Texas Health San Antonio (UT Health) Institutional Animal Care and Use Committee, Protocol #20220056AR.