Fig 1.
CCC-003 specifically recognizes the DNA sequence of mutant ALK.
(A) Chemical structure of CCC-003, a sequence-specific DNA-alkylating agent directly targeting the F1174L-mutated ALK gene, and the mismatch polyamide (Mismatch), which had no binding motif within the coding region of the ALK gene. The site of alkylation is shown in red. (B) Schematic drawing of CCC-003 and Mismatch at the site of ALK F1174L mutation. Blue open circles represent pyrrole moieties and red circles represent imidazole. Arrow indicates the position of the ALK F1174L mutation. (C) SPR sensorgrams for the interaction of CCC-003-Dp with hairpin DNAs containing the F1174L mutation sequence, Mismatch-Dp with hairpin DNAs containing the F1174L mutation sequence, CCC-003-Dp with hairpin DNAs containing the wild-type sequence, and Mismatch-Dp with hairpin DNAs containing the wild-type sequence. Oligonucleotides were immobilized on the surface of an SA sensor chip. Five curves of the lowest, mid-low, middle, mid-high, and highest concentration of PI polyamide indicate 100, 200, 300, 400, and 500 nM, respectively. (D) Oligonucleotides used in PAGE analysis. (E) PAGE analysis confirmed the alkylation site on target DNA fragment by CCC-003. Annealed DNA fragment (dsDNA) using 5′-TAMRA-labeled DNA oligonucleotides (ODN1 and ODN2) was incubated with DMSO, Mismatch polyamide or CCC-003 and heated to visualize alkylated bands by PAGE analysis. The alkylated DNA fragments were analyzed with reference DNAs (ODN3 and ODN4).
Table 1.
Binding affinities of PI polyamides for ALK mutation (F1174L) or ALK wild-type (WT) oligonucleotides.
Fig 2.
CCC-003 inhibits ALK expression in ALK-mutated neuroblastoma cell lines.
(A) Western blotting analysis confirmed the expressions of wild-type (WT) and F1174L-mutated ALK in Ba/F3 cells after G418 selection. (B) WST assay in ALK-expressing Ba/F3 cells. The cells were cultured with the indicated concentrations of IL-3 for 72 h. (C) Ba/F3 cells expressing ALK were treated with 0.01–100 nM CCC-003 in the absence of IL-3 for 72 h and subjected to WST assay. (D) WST assay in neuroblastoma cells. SK-N-AS, SK-N-FI, SH-SY5Y, and Kelly cells were incubated with the indicated concentrations of CCC-003. 72 h after treatment, the percentage of viable cells was determined and depicted using GraphPad Prism. Error bars indicate the SD of data from triplicate experiments. (E) ALK wild-type SK-N-AS and ALK F1174L-mutated SH-SY5Y and Kelly cells were treated with DMSO for 24 h. ALK mRNA expression was analyzed using qRT-PCR. RPS18 was used as an internal control. P-values were determined using one-way ANOVA followed by a Bonferroni post-hoc test (***, P < 0.001). Data are presented as the mean ± SD from three independent experiments. (F) SK-N-AS, SH-SY5Y, and Kelly cells were treated with 3–30, 1–10, or 10–100 nM CCC-003 for 24 h, respectively. ALK mRNA expression was analyzed using qRT-PCR. DMSO was used as a control treatment. RPS18 expression was used as a housekeeping gene. P-values were determined using one-way ANOVA followed by a Bonferroni post-hoc test (**, P < 0.01; ***, P < 0.001). Data are presented as the mean ± SD from three independent experiments. (G) SK-N-AS and SH-SY5Y cells were treated with 1–10 nM CCC-003, and Kelly cells were treated with 30–100 nM CCC-003 for 48 h. ALK protein levels were analyzed using western blotting.
Fig 3.
CCC-003 inhibits cell proliferation and ALK expression in ALK-mutated neuroblastoma cell lines to a greater extent than other ALK inhibitors.
(A, B, C) WST assay in ALK wild-type and ALK-mutated cells. (A) SK-N-AS cells were incubated for 72 h after treatment with CCC-003 or ALK inhibitors crizotinib and alectinib. (B) SH-SY5Y cells were incubated for 72 h after treatment with CCC-003 or ALK inhibitors. (C) Kelly cells were incubated for 72 h after treatment with CCC-003 or ALK inhibitors. (D) IC50 values of CCC-003, crizotinib, and alectinib. (E) SH-SY5Y cells were treated with 3–10 nM CCC-003 or 100 nM ALK inhibitors for 72 h. ALK and pALK protein levels were determined using western blotting analysis.
Fig 4.
CCC-003 specifically suppresses ALK expression in ALK-mutated neuroblastoma cell lines.
(A) WST assay in ALK wild-type and ALK-mutated cells. SK-N-AS, SH-SY5Y and Kelly cells were incubated for 72 h after treatment with CCC-003 or mismatch polyamide. (B) SH-SY5Y cells were treated with 3 nM CCC-003 or mismatch polyamide, and Kelly cells were treated with 30 nM CCC-003 or mismatch polyamide for 24 h. ALK mRNA expression was analyzed using qRT-PCR. P-values were determined using one-way ANOVA followed by a Bonferroni post-hoc test (**, P < 0.01; ***, P < 0.001). (C) SH-SY5Y cells were treated with 3 nM CCC-003, and Kelly cells were treated with 30 nM CCC-003 for 48 h. ALK, pALK, Shc, and pShc protein levels were determined using western blotting.
Fig 5.
CCC-003 induces apoptotic cell death in ALK-mutated neuroblastoma cell lines.
(A, B) Flow cytometric analysis by Annexin V staining of neuroblastoma cells. P-values were determined using one-way ANOVA followed by a Bonferroni post-hoc test (***, P < 0.001). (A) SK-N-AS, SH-SY5Y, and Kelly cells were treated with 1–100, 1–10, and 10–100 nM CCC-003 for 48 h, respectively. Representative images of Annexin V-positive cells detected using flow cytometry. (B) SH-SY5Y cells were treated with 3 nM CCC-003 or mismatch polyamide, and Kelly cells were treated with 30 nM CCC-003 or mismatch polyamide for 48 h. (C) SH-SY5Y cells were treated with 3 nM CCC-003 or mismatch polyamide, and Kelly cells were treated with 30 nM CCC-003 or mismatch polyamide for 48 h. Protein expression of cleaved PARP and cleaved caspase-3 was determined using western blotting.
Fig 6.
CCC-003 suppresses tumor growth in human ALK-mutated neuroblastoma xenograft models.
(A, B, C) SH-SY5Y cells were subcutaneously injected into the flanks of female immune-deficient BALB/c nu/nu mice. Administration of CCC-003 began when the average tumor size reached 500 mm3. (A) The tumor volume of mice was measured twice a week. Red arrows indicate the timepoints of CCC-003 administration. P-values were determined using repeated measures ANOVA followed by a Bonferroni post-hoc test (*, P < 0.05). Data are presented as the mean ± SD. DMSO was used as a control. (B) Representative images of mice with SH-SY5Y xenografts at 24 h after administration. (C) Representative images of SH-SY5Y xenograft tumors at 24 h after administration. At 24 h after administration, the tumor tissues were collected and used for immunohistochemistry with HE and an anti-cleaved caspase-3 antibody. Scale bars, 50 μm. (D) The number of cleaved caspase-3-positive cells was determined in tumor tissues. P-values were determined using a paired t-test (***, P < 0.001).