Abstract Objective: We aimed to model the evolution of chemoresistance to methotrexate, doxorubicin, and cisplatin (MAP) in vitro and to characterize collateral drug sensitivity and resistance patterns over time, potentially informing second-line treatment strategies in chemo resistant primary or recurrent disease. Methods: Five evolutionary replicates of MG63.3 osteosarcoma cells were treated with six complete cycles of pulsed, clinically relevant doses of cisplatin and doxorubicin, alternating with methotrexate. Dose-response curves were performed after each chemotherapy cycle to assess resistance to MAP and collateral responses to 16 drugs and combinations. RNA sequencing and transcription factor activity inference were performed across treatment timepoints to temporally explore transcriptional correlates of drug response. Results: Cells developed resistance to doxorubicin (1.49-2.59 fold) and methotrexate (0.92-2.87 fold) but remained sensitive to cisplatin. Collateral resistance emerged to etoposide, vincristine, and topotecan, while sensitivity increased to gemcitabine, cabozantinib, and palifosfamide. Collateral responses varied temporally, with some increasing linearly with evolution of MAP resistance, and some appearing transient or stochastic. Transcriptional profiling revealed increased activity of E2F and ERG and context-dependent roles for interferon signaling. Resistance phenotypes persisted after drug withdrawal and cryopreservation. Conclusion: Our model reveals temporally dynamic, heterogeneous collateral responses during the development of chemoresistance to MAP in osteosarcoma. While cross-resistance to several agents emerged, collateral sensitivity to gemcitabine and cabozantinib supports a second line use for these agents. These data may inform rational sequencing of therapies to preempt or exploit resistance evolution. Citation Format: Elizabeth Nowak, Jarrell Imamura, Arda Durmaz, Masahiro Hitomi, Zachary Burke, Jacob G. Scott. Evolving toward precision oncology: Leveraging collateral drug responses for personalized second-line osteosarcoma treatment 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 3542.
Nowak et al. (Fri,) studied this question.