ARTICLE Lineage-defined leiomyosarcoma subtypes emerge years before diagnosis and determine patient survival Nathaniel D. Anderson 1,2 , Yael Babichev 3,17 , Fabio Fuligni 4,17 , Federico Comitani 1 , Mehdi Layeghifard 1 , Rosemarie E. Venier 3 , Stefan C. Dentro 5 , Anant Maheshwari 1 , Sheena Guram 3 , Claire Wunker 3,6 , J. Drew Thompson 1 , Kyoko E. Yuki 1 , Huayun Hou 1 , Matthew Zatzman 1,2 , Nicholas Light 1,6 , Marcus Q. Bernardini 7,8 , Jay S. Wunder 3,9,10 , Irene L. Andrulis 2,3,11 , Peter Ferguson 7,9,10 , Albiruni R. Abdul Razak 7 , Carol J. Swallow 3,6,9,12 , James J. Dowling 1,11 , Rima S. Al-Awar 13,14 , Richard Marcellus 13 , Marjan Rouzbahman 2,7 , Moritz Gerstung 5 , Daniel Durocher 3,11 , Ludmil B. Alexandrov 15 , Brendan C. Dickson 2,3,16 , Rebecca A. Gladdy 3,6,9,12 ✉ & Adam Shlien 1,2,4 ✉ Leiomyosarcomas (LMS) are genetically heterogeneous tumors differentiating along smooth muscle lines. Currently, LMS treatment is not informed by molecular subtyping and is associated with highly variable survival. While disease site continues to dictate clinical management, the contribution of genetic factors to LMS subtype, origins, and timing are unknown. Here we analyze 70 genomes and 130 transcriptomes of LMS, including multiple tumor regions and paired metastases. Molecular profiling highlight the very early origins of LMS. We uncover three specific subtypes of LMS that likely develop from distinct lineages of smooth muscle cells. Of these, dedifferentiated LMS with high immune infiltration and tumors primarily of gynecological origin harbor genomic dystrophin deletions and/or loss of dystrophin expression, acquire the highest burden of genomic mutation, and are associated with worse survival. Homologous recombination defects lead to genome-wide mutational signatures, and a corresponding sen- sitivity to PARP trappers and other DNA damage response inhibitors, suggesting a promising therapeutic strategy for LMS. Finally, by phylogenetic reconstruction, we present evidence that clones seeding lethal metastases arise decades prior to LMS diagnosis. https://doi.org/10.1038/s41467-021-24677-6 OPEN 1 Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada. 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. 3 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. 4 Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, ON Ontario, Canada. 5 European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, UK. 6 Institute of Medical Science, University of Toronto, Toronto, ON, Canada. 7 Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada. 8 Department of Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada. 9 Department of Surgery, University of Toronto, Toronto, ON, Canada. 10 University Musculoskeletal Oncology Unit, Mount Sinai Hospital, Toronto, ON, Canada. 11 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 12 Division of General Surgery, Mount Sinai Hospital, Toronto, ON, Canada. 13 Drug Discovery Program, Ontario Institute for Cancer Research, Toronto, ON, Canada. 14 Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada. 15 Department of Cellular and Molecular Medicine and Department of Bioengineering and Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA. 16 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada. 17 These authors contributed equally: Yael Babichev, Fabio Fuligni. ✉ email: gladdy@lunenfeld.ca; adam.shlien@sickkids.ca NATURE COMMUNICATIONS | (2021)12:4496 | https://doi.org/10.1038/s41467-021-24677-6 | www.nature.com/naturecommunications 1 1234567890():,;