Mouse models

All procedures involving mice were guided by the DFCI Animal Care and Use Committee and performed under an IRB-approved protocol. All mouse experiments were performed using subcutaneous injections of 1×10⁶ cells into 4–6-week-old recipient female mice, with littermates randomly assigned to experimental groups. NB-9464 TH-MYCN murine neuroblastoma xenografts were generated in syngeneic C57BL/6J mice, while human neuroblastoma patient-derived (COG-N-415x) xenografts were generated in nude mice (NU/J). For the first MTD study, C57BL/6J mice were treated with CMLD012824 (0.1, 0.2, 0.4 mg/kg) diluted in solvent (5.2% PEG300, 5.2% Tween-80) daily for 7 days by intraperitoneal injection. After reaching assay endpoint of 12 days, livers were excised from vehicle-treated and CMLD012824 -treated (1 mg/kg) animals for WB analysis. For the second MTD study, C57BL/6J mice bearing NB-9464 tumors were randomly assigned into groups upon tumor volume reaching 100–200 mm³, with the volume being approximately equal between groups and treated with CMLD012824 (0.1, 0.2 mg/kg) diluted in solvent (5.2% PEG300, 5.2% Tween-80) three times per week for 30 days by intraperitoneal injection. For the efficacy study, nude mice bearing COG-N-415x tumors were randomly assigned to treatment groups upon tumor volume reaching 100–200 mm³ and treated with 0.2 mg/kg CMLD012824 (diluted in 5.2% PEG300, 5.2% Tween-80) or vehicle control (DMSO in 5.2% PEG300, 5.2% Tween-80) three times per week for 40 days by intraperitoneal injection. Tumor size and body weight were monitored three times per week and tumor volume was calculated using the ellipsoid formula 1/2(max diameter x min diameter²). Once tumors reached 1000 mm³, the mice were euthanized according to approved animal protocols. Tumors were either fixed in 10% neutral buffered formalin, or snap frozen and stored at −80°C until further analysis. All animal experiments were conducted according to approved protocols by IACUC.