(P 260) MECHANISM AND EFFICACY OF ANTHRACYCLINE MEDIATED EPIGENETIC DISRUPTION IN BONE SARCOMAS.

Objective: Doxorubicin (Doxo) is a cornerstone therapy for osteosarcoma (OS) and Ewing sarcoma (EwS), but its use is limited by severe off-target toxicities. In addition to Topo II–mediated genotoxicity, Doxo disrupts chromatin through a Topo II–independent mechanism. Chemical modification of Doxo enables separation of chromatin-disrupting effects from DNA damage. Here, we investigate dimethyl-doxorubicin (DMD), a novel anthracycline analog that retains anti-tumor potency with reduced toxicity. We assess its preclinical efficacy, safety, and elucidate its epigenetic consequences in bone sarcoma models.

Methods: Doxo and DMD efficacy was assessed in EwS and OS cell lines, PDX models, and immunocompromised in vivo xenografts. Doxo-pretreated C57BL/6 mice were used to mimic DMD safety in the relapsed setting, with survival and toxicity monitored for 12 weeks. CUT&RUN assessed effects on targeted histone marks (H3K27ac, H3K4me3, H3K27me3, H3K4me1), transcription factors (EWS::FLI1, MYC), and RNA pol II. Multiplexed Assay for the Rational Modulation Of Transcription factors (MARMOT), a novel mass spectrometry-based assay, was used to comprehensively profile protein-chromatin interactions across the genome.

Results: DMD induced apoptosis comparable to Doxo in established and PDX-derived OS and EwS cell lines at nanomolar concentrations in vitro, without evidence of DNA damage. In vivo, DMD suppressed tumor growth equivalently to Doxo in an EwS xenograft model; OS PDX studies are ongoing. C57BL/6 mice with prior Doxo exposure showed improved tolerability to subsequent DMD treatment compared to repeated Doxo administration. CUT&RUN profiling revealed that both agents potently disrupted chromatin binding of H3K27ac, H3K4me3 key, EWS::FLI1, MYC, and RNA pol II, without altering total protein levels. Bulk RNA-seq studies are ongoing to assess downstream transcriptional consequence. Using the MARMOT platform we found that anthracyclines globally perturb protein–chromatin interactions. Notably, a subset of ribosomal proteins was enriched following treatment, suggesting that co-targeting ribosome biogenesis may potentiate DMD efficacy.

Conclusion: Our studies show that DMD is a safe and potent anthracycline for bone sarcomas, inducing tumor cell death without significant DNA damage. It broadly disrupts protein–chromatin interactions and reveals ribosome biogenesis as a potential co-targetable vulnerability. These findings support further development of DMD as a therapeutic backbone for bone sarcomas.