Disrupting YAP1-mediated glutamine metabolism induces synthetic lethality alongside ODC1 inhibition in osteosarcoma

Purpose: Osteosarcoma, a highly aggressive primary bone cancer predominantly affecting adolescents, often becomes resistant to initial chemotherapy, leading to metastasis and limited treatment options. This study aims to identify novel therapeutic targets for metastatic and recurrent osteosarcoma.

Methods: We investigated the potential of targeting the YAP1-regulated glutamine metabolic pathway to enhance the response of osteosarcoma (OS) to DFMO. We began by analyzing single-cell transcriptomic data to assess the activation of polyamine metabolism in OS patients with MTAP deletions. This was supported by transcriptome sequencing data from recurrent and non-recurrent OS tissues, which confirmed that polyamine metabolism is activated in progressive OS. High-throughput drug screening identified CIL56, a YAP1 inhibitor, as a promising candidate for combination therapy with DFMO. We validated this drug combination’s efficacy using PDX and CDX models in vivo. In vitro, we performed western blot analysis, qPCR, immunofluorescence staining, and PuMA experiments to track changes in molecular expression, distribution, and tumor metastasis capabilities. Proliferation was assessed using CCK-8 and colony formation assays. We used flow cytometry and reactive oxygen species (ROS) probes to observe changes in ROS levels and glutamine metabolism. RNA-seq and metabolomics were applied to identify metabolic changes in OS cells treated with the DFMO and CIL56 combination, allowing us to validate the role of the YAP1-mediated glutamine metabolic pathway in DFMO resistance.

Results: Analysis of single-cell RNA-seq data revealed a subset of late-stage OS cells with significantly increased polyamine metabolism. This finding was further confirmed by transcriptomic profiling of recurrent and non-recurrent OS tissues. High-throughput screening highlighted a promising combination of DFMO and CIL56. DFMO treatment reduced YAP1 protein phosphorylation in OS cells, leading to increased nuclear entry and activation of the YAP1-regulated glutamine metabolic pathway. This activation lowered intracellular ROS levels, which counteracts DFMO’s anticancer effects. Combining DFMO with the YAP1 inhibitor CIL56 or the glutaminase inhibitor CB-839 significantly enhanced therapeutic efficacy both in vivo and in vitro, emphasizing the potential of targeting the YAP1-mediated glutamine metabolic pathway to improve DFMO’s effectiveness.

Conclusion: Our study demonstrates that YAP1-mediated glutamine metabolism acts as a critical escape mechanism against DFMO, following polyamine metabolism inhibition. These findings offer valuable insights into the potential of DFMO in a “one-two punch” therapy for metastatic and recurrent osteosarcoma.