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Supplementary data including supplementary figures S1 to S6 and supplementary tables S1 to S3.

Supplementary information on the used materials and methods.

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2–dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2–dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2–dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2–dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax–idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.

Purpose: Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. Although patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA deficiency and to test new combination therapies to establish optimal treatment strategies.Experimental Design: We did a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors.Results: Three alkylators—chlorambucil, melphalan, and nimustine—displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine doing better than cisplatin and the poly-(ADP-ribose)-polymerase inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 of 5 animals tested.Conclusions: We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators, especially melphalan and nimustine, alone or in combination with the poly-(ADP-ribose)-polymerase inhibitors, for the treatment of breast cancers with a defective BRCA pathway. Clin Cancer Res; 16(1); 99–108