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. 2018 Jun;17(6):1167-1176.
doi: 10.1158/1535-7163.MCT-17-0834. Epub 2018 Apr 6.

APTO-253 Is a New Addition to the Repertoire of Drugs that Can Exploit DNA BRCA1/2 Deficiency

Cheng-Yu Tsai  1 , Si Sun  1 , Hongying Zhang  2 , Andrea Local  2 , Yongxuan Su  3 , Larry A Gross  3 , William G Rice  2 , Stephen B Howell  4   5
Affiliations

APTO-253 Is a New Addition to the Repertoire of Drugs that Can Exploit DNA BRCA1/2 Deficiency

Cheng-Yu Tsai et al. Mol Cancer Ther. 2018 Jun.

Abstract

APTO-253 is a small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies. We sought to determine the mechanisms of action and basis for resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination studies. The cellular pharmacology of APTO-253 was analyzed in Raji lymphoma cells and a subline selected for resistance (Raji/253R). Using LC/MS/ESI analysis, APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. The intracellular content of Fe(253)3 exceeded that of the native drug by approximately 18-fold, and Fe(253)3 appears to be the most active form. Treatment of cells with APTO-253 caused DNA damage, which led us to ask whether cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug. It was found that loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 of a magnitude similar to the effects of PARP inhibitors, olaparib. Raji cells selected for 16-fold acquired resistance had 16-fold reduced accumulation of Fe(253)3 RNA-seq analysis revealed that overexpression of the ABCG2 drug efflux pump is a key mechanism of resistance. ABCG2-overexpressed HEK-293 cells were resistant to APTO-253, and inhibition of ABCG2 reversed resistance to APTO-253 in Raji/253R. APTO-253 joins the limited repertoire of drugs that can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. Mol Cancer Ther; 17(6); 1167-76. ©2018 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

H. Zhang, A. Local, and W.G. Rice are employees of Aptose Biosciences, Inc. S.B. Howell is a consultant of Aptose Biosciences. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1.
Figure 1.
Fe(253)3 is an active intracellular form of APTO-253. A, Structure of APTO-253. B, Structure of Fe(253)3. C, Relative cytotoxicity of APTO-253 (●) and Fe(253)3 (■) in the Raji cells. D, The intracellular accumulation of APTO-253 (■) and Fe(253)3 (■) in Raji cells exposed to 0.5 μmol/L APTO-253 or Fe(253)3 for 6 hours. Vertical bars, ±SEM, where missing SEM is less than the size of the symbol. ***, P < 0.001; ****, P < 0.0001.
Figure 2.
Figure 2.
APTO-253 causes DNA damage. A, The accumulation of phospho-ATM, phospho-H2AFX, and cleaved PARP in the Raji cells as a function of duration of exposure to 0.5 μmol/L APTO-253. The immunoblot shown is a representative of three independent experiments. B, Representative immunofluorescent images of nuclear foci formation comparing DMSO- and APTO-253–treated CAOV3 cells. C, Mean ± SEM number of γ-H2AFX foci per cell; n = 100. D, Box and whisker plot showing neutral comet assay quantification of percent tail DNA in Raji cells treated with DMSO or 0.5 μmol/L APTO-253 for 6 hours. n = number of cells examined; vertical bars, ±SEM. ****, P < 0.0001.
Figure 3.
Figure 3.
Loss of BRCA1 and BRCA2 function results in hypersensitivity to APTO-253. Sensitivity of BRCA1-proficient and -deficient isogenic MCF10A clones (A), hTERT-IMEC clones (B), and MCF7 (C) to olaparib (top) and APTO-253 (bottom). Sensitivity of BRCA2-proficient and -deficient isogenic PEO4 and PEO1 (D), and HCT116 BRCA2-deficient clones (E) to olaparib (top) and APTO-253 (bottom). The accumulation of γ-H2AFX in the MCF7 control and shBRCA1 clone E7 cells (F) and the BRCA2-proficient HCT116 and the deficient clone B18 cells treated with DMSO or the indicated concentration of APTO-253 for 24 hours (G). Vertical bars, ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4.
Figure 4.
Characterization of cells resistant to APTO-253. A, Concentration–survival curves for Raji (formula image), Raji/253R (formula image), Raji/253R, and Raji/253R cells after culture in drug-free medium for 3 months (formula image). B, Western blot analysis of proteins involved in apoptosis in Raji and Raji/253R treated with DMSO or APTO-253 0.5 μmol/L for 24 hours. C, The intracellular accumulation of APTO-253 (formula image) and Fe(253)3 (formula image) in Raji and Raji/253R cells after a 6-hour exposure to 0.5 μmol/L APTO-253. D, The intracellular accumulation of Fe(253)3 in the Raji and Raji/253R cells at 6 hours as a function of APTO-253 concentration. Vertical bars, ±SEM. ** P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 5.
Figure 5.
Role of ABCG2 in resistance to APTO-253. A, Relative levels of ABCG2 mRNA in Raji and Raji/253R. B, Western blots of biotinylated proteins were probed with anti-ABCG2 antibody. ATP1A1 served as a loading control. C, Cytotoxicity of Ko143 in Raji (formula image) and Raji/253R (formula image). D, Concentration–survival curves for Raji (formula image) and Raji/253R (formula image) treated with APTO-253 alone or in combination with APTO-253 and 5 nmol/L (formula image) or 50 nmol/L Kol43 (formula image). E, Cytotoxicity of topotecan in Raji (formula image) and Raji/253R (formula image) and the combination of topotecan and 50 nmol/L Ko143 in Raji/253R (formula image). F, Cytotoxicity of carboplatin in Raji (formula image) and Raji/253R (formula image) and the combination of carboplatin and 50 nmol/L Ko143 in Raji/253R (formula image). G, Concentration–survival curves for HEK-293 transfected with pcDNA (formula image) and ABCG2, clone R5 (formula image) treated with APTO-253. Vertical bars, ± SEM. **, P < 0.01; **, P < 0.001.
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