Combination treatment with mitoxantrone and doxorubicin induces multidrug resistance in Ewing Sarcoma CDX reporter xenografts.

Ewing Sarcoma (ES) is an aggressive malignancy with limited treatment options in advanced disease. This study aimed to identify efficacious therapeutic candidates from FDA-approved drugs via in silico screening and evaluate the potential of the top candidate, mitoxantrone (Mito), alone and in combination with standard-of-care doxorubicin (Doxo). In silico screening was performed against an ES-specific molecular expression profile. Mito was validated in ES in vitro cellular models, and combination effects with Doxo were assessed by cytotoxicity assays. In vivo efficacy was evaluated in ES cell-line-derived xenograft (CDX) reporter models, with MDR1 (ABCB1) expression quantified across treatment arms to assess multidrug resistance. Mito demonstrated significant in vitro cytotoxicity, and Mito + Doxo produced an additive effect. However, in vivo CDX models showed no significant tumor response improvement with combination treatment relative to monotherapies. Combination-treated xenografts showed increased tumorigenicity, while monotherapy arms showed significant antitumor efficacy. MDR1 expression was markedly elevated in combination-treated tumors, indicating an acquired multidrug-resistant phenotype. This study reveals a critical in vitro and in vivo discordance and suggests that Mito + Doxo co-administration may paradoxically induce multidrug resistance, abrogating backbone chemotherapy efficacy. These findings urge caution in preclinical combination strategies involving standard-of-care agents and underscore the need for further investigation prior to clinical translation in ES.