Abstract Metastasis accounts for most cancer related morbidity and mortality and is strongly influenced by host genetic background. Using inbred mouse strains, germline single nucleotide polymorphisms (SNP) can shape the tumor microenvironment, immune response, and systemic physiology to permit or restrict metastatic spread. Given the central role of mitochondria in regulating metabolism and cellular signaling, mitochondrial genetics represent an additional, and largely unexplored, dimension of host control over metastasis. To isolate mitochondrial from nuclear contributions, we compared wild-type to mitochondrial nuclear exchange (MNX) mice, in which nuclear genomes from one inbred strain are paired with mitochondrial genomes from another. Across multiple oncogenic drivers, mtDNA significantly alters primary tumor growth and metastatic efficiency. No broad-scope alterations in metabolism distinguish MNX from nuclear genome-matched wild-type mice. So, we focused on a SNP localized to the mitochondrial tRNA for arginine (mt-TR), where distinct SNP correlate with differences in metastasis, nuclear gene regulation, immune profiles, and gut microbial composition. Since nucleus encoded tRNA regulate diverse cellular processes (including transcription, translation, stress responses, and metastasis) via formation of tRNA-derived fragments (tRF), we hypothesized that mitochondrial tRF can function similarly. Using northern blotting, we identified previously unreported mt-TR-derived tRF and found that their expression varies by SNP, sex, and tissues. The fragment patterns indicate that mt-TR undergoes defined site cleavage rather than random degradation. We deduced that tRF derived from mt-TR are highly modified molecules with terminal end modifications and/or base methylations that prevent detection by standard small RNA sequencing. After enzymatic removal of these modifications, the fragments were sequenced, allowing us to map cleavage sites and resolve SNP-dependent fragment populations. These molecules constitute some of the earliest evidence that mitochondrially encoded tRF may contribute to the regulation of complex cancer phenotypes such as metastasis. Ongoing work will determine the molecular interactors which will define molecular networks influenced by mt-tRF. This work has the potential to identify previously unrecognized pathways and metastasis-regulatory mechanisms that can be leveraged to identify new targets for cancer therapy. Support: Metavivor Inc., Natl. Fndn. Cancer Res., Allison’s Allies, Hall Family Fndn., P30-CA168524 Citation Format: Katy L. Swancutt, Sydney Quijano, Gabrielle Fisette, Emily Schueddig, Devin C. Koestler, Isidore Rigoutsos, Yi Jing, Danny R. Welch. Mitochondrial tRNA fragments as modifiers of breast cancer development and metastasis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1368.
Swancutt et al. (Fri,) studied this question.