AbstractAlthough great advances have been made over the past decades, therapeutics for osteosarcoma are quite limited. We performed long‐read RNA sequencing and tandem mass tag (TMT)‐based quantitative proteome on osteosarcoma and the adjacent normal tissues, next‐generation sequencing (NGS) on paired osteosarcoma samples before and after neoadjuvant chemotherapy (NACT), and high‐throughput drug combination screen on osteosarcoma cell lines. Single‐cell RNA sequencing data were analyzed to reveal the heterogeneity of potential therapeutic target genes. Additionally, we clarified the synergistic mechanisms of doxorubicin (DOX) and HDACs inhibitors for osteosarcoma treatment. Consequently, we identified 2535 osteosarcoma‐specific genes and several alternative splicing (AS) events with osteosarcoma specificity and/or patient heterogeneity. Hundreds of potential therapeutic targets were identified among them, which showed the core regulatory roles in osteosarcoma. We also identified 215 inhibitory drugs and 236 synergistic drug combinations for osteosarcoma treatment. More interestingly, the multiomic analysis pointed out the pivotal role of HDAC1 and TOP2A in osteosarcoma. HDAC inhibitors synergized with DOX to suppress osteosarcoma both in vitro and in vivo. Mechanistically, HDAC inhibitors synergized with DOX by downregulating SP1 to transcriptionally modulate TOP2A expression. This study provided a comprehensive view of molecular features, therapeutic targets, and synergistic drug combinations for osteosarcoma.