APTO-253 is a small molecule with anti-proliferative activity against cell lines derived from a wide range of human malignancies. The goal of this project was to investigate the mechanism of action so as to identify synthetic lethal interactions that can guide combination drug studies. APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3] using LC/MS/ESI analysis. The intracellular content of Fe(253)3 exceeded that of the native drug by ~18-fold, and Fe(253)3 appears to be the the most active form. The structure of Fe(253)3 is similar to drugs that stabilize quadruplex DNA (G4) structures, and Fe(253)3 was found to stabilize G4s in vitro and to reduce the expression of Myc. Stabilization of G4 structures has been reported to produce DNA strand breaks, which led us to investigate whether APTO-253 caused DNA damage. Indeed, treatment with APTO-253 produced time and concentration-dependent γH2AX foci formation in an array of diverse cell lines. APTO-253 was also found to produce DNA double strand breaks, as detected by the neutral comet assay, which led us to ask if cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug in a manner similar to the effects of PARP inhibitors such as olapirib. BRCA1-deficient sublines of 3 isogenic pairs of cell lines (MCF10A, MCF7 and hTERT-IMEC), and BRCA2-deficient sublines of 2 isogenic pairs (PEO1, HCT116/B18 and HCT116/B43) were all hypersensitive to olaparib and APTO-253. Thus, loss of either BRCA1 or BRCA2 function renders malignant cells hypersensitive to APTO-253. When parental HCT116 and the BRCA2-deficient B18 subline were treated with APTO-253 for 24 h, there were significant increases in γH2AX and cleaved PARP only in B18 clone, suggesting BRCA2 is required to repair DNA damage generated by APTO-253. We report here that APTO-253 is converted intracellularly into an Fe complex, and that this Fe(253)3 complex is likely the active form of the drug. APTO-253 stabilizes certain quadruplex DNA structures, causes DNA damage, and exhibits synthetic lethality comparable to olaparib, albeit through a different mechanism, in cells deficient in either BRCA1 or BRCA2 function. Unlike other drugs for which loss of this repair function results in hypersensitivity, APTO-253 does not produce myelosuppression even at the maximum tolerated dose. The observations reported here also identify γH2AX as a potential biomarker of clinical effect and open the window more detailed studies of how APTO-253 promotes DNA damage and how it might be used clinically to treat patients with tumors harboring deficiencies in DNA repair.

Citation Format: Cheng-Yu Tsai, Si Sun, Hongying Zhang, Andrea Local, William Rice, Stephen B. Howell. APTO-253 is a new addition to the repertoire of drugs that can exploit DNA BRCA1/2 deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4840.