Mouse experiments.

All mouse experiments were performed with approval from the Institutional Animal Care and Use Committee (IACUC) of the DFCI. Three mouse experiments were performed: (i) to assess the tumorigenic potential of resistant cells in vivo; (ii) to assess that resistance to TAE684 was maintained in vivo; and (iii) to assess the effect of JQ1 on resistant cells in vivo. All experiments were performed using subcutaneous cell xenograft models generated by injecting 2 × 106 sensitive or resistant Kelly neuroblastoma cells into the flanks of NU/NU (Crl:NU-Foxn1^nu) (Charles River Laboratories) or NU/NU (CrTac:NCr-Foxn1^nu) (Taconic) 7-week-old female mice. Mice were randomized into groups of equal average volumes, and investigators were not blinded to group allocation during data collection. (i) To assess the tumorigenic potential of resistant cells in absence of treatment, mice with established disease (mean tumour volume of 200 mm3) were monitored for up to 23 days (n = 4 per group). Tumours were obtained, dissociated and used to establish cell lines and for assessment of mRNA levels, protein expression and sensitivity to TAE684. (ii) To ensure that the in vitro resistance to TAE684 was maintained in vivo, mice with established disease were divided into two cohorts and were treated with either TAE684 (10 mg kg⁻¹) or vehicle control by oral gavage once daily (n = 8 per group), and were monitored for up to 56 days from start of treatment. (iii) To assess the sensitivity of resistant cells to BRD4 inhibition, mice with established disease were divided into two cohorts and treated with either JQ1 (50 mg kg⁻¹) or vehicle control intraperitoneally (i.p.) once daily (n = 6 per group), and were monitored for up to 87 days from start of treatment. For all experiments, disease burden was quantified using electronic caliper measurements (2–3 times a week) and mouse weights were monitored at least twice a week. Tumour volumes were calculated using the modified ellipsoid formula⁴¹: ½(length × width2). Animals were euthanized when tumour volumes reached 1,500–2,000 mm3 based on institutional IACUC criteria for maximum tumour volumes. In none of the experiments were the institutional limits for tumour volumes (<2,000 mm3 measurement preceding the day of euthanization) exceeded.