Abstract B026: The genomic landscape of canine osteosarcoma implicates DMD as a therapeutic vulnerability
Authors: Gardner Heather L. , Sivaprakasam Karthigayini , Briones Natalia , Hendricks William , London Cheryl A. 
Published: March 2020

Abstract
Background: Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in outcome: 30% of people and 90% of dogs still die of metastasis. While the genomic landscape of human OS has been interrogated, limited data exist regarding that of dogs. Using whole genome sequencing (WGS) we identified the presence of mutations in DMD, the gene encoding dystrophin, in canine OS. The purpose of this work was to expand upon these findings and begin to interrogate the functional consequences of these mutations. Methods: Whole-genome sequencing (WGS) of 24 canine primary OS tumor samples was completed on an Illumina platform. Somatic single nucleotide and structural variants, and germline variants were identified using Cerberus, a canine cancer genomics pipeline. Expression of the Dp71 DMD isoform was modulated using CRISPR-Cas9 and lentiviral overexpression approaches; the biologic significance of DMD loss was characterized using Co-IP, western blotting, Matrigel invasion and proliferation assays. Results: Our prior data found that canine OS recapitulates several genetic features of human OS including high structural complexity and translocation burden withTP53 (71%) the most frequently mutated gene. In addition to inactivating SETD2 mutations 42% of cases, we found that 50% of tumors possessed large somatic intragenic deletions and copy number loss of DMD, the gene encoding dystrophin. Building upon these data we found that truncated dystrophin isoforms (Dp260, Dp140, Dp71, Dp40) predominate in canine OS cell lines, with only Dp40 expressed in normal osteoblasts. Deletion of Dp71 in OS lines using CRISPR-Cas9 resulted in increased cell proliferation and markedly enhanced invasion through Matrigel. Consistent with the published literature, dystrophin and β-dystroglycan co-associated in the OS lines, and phosphorylation of β-dystroglycan was increased when dystrophin expression was downregulated. Lastly, dasatinib, a small molecule inhibitor of Src, inhibited β-dystroglycan phosphorylation and cell proliferation in the canine OS lines. Conclusions: These data provide important new information regarding the genomic profile of canine OS and suggest that aberrations in dystrophin expression may contribute to OS pathophysiology in dogs by promoting a metastatic phenotype. Future work will dissect exactly how altered dystrophin isoforms contribute to OS tumor biology with the goal of developing approaches for therapeutic intervention that target this pathway.
Citation Format: Heather L. Gardner, Karthigayini Sivaprakasam, Natalia Briones, William Hendricks, Cheryl A. London. The genomic landscape of canine osteosarcoma implicates DMD as a therapeutic vulnerability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B026. doi:10.1158/1535-7163.TARG-19-B026