Figure 1.
SYC-522 decreased the methylation of H3K79 in MLL-rearranged leukemia cell lines.
(A) MV4-11 cells were treated with 3 µM SYC-522 and (B) MOLM13 cells were treated with 10 µM SYC-522. The histone proteins were extracted from the cell lysates after 0, 3, or 6 days of treatment. H3K79 di-methylation, H3K4 tri-methylation and H3K27 tri-methylation were assessed using specific antibodies. Representative Western blots are shown on the left, and averaged densitometry values are shown on the right. Levels of methylated H3K4, H3K27 and H3K79 were normalized to the corresponding total H3 and shown as a fold change of values for untreated cells. Bars represent the mean ±SEM of 3 independent experiments. *: p<0.05, day 0 vs. day 3 or 6, unpaired t-test.
Figure 2.
SYC-522 decreased the expression of key genes in leukemia cell lines.
HOXA9, MEIS1, CCND1 and BCL2L1 mRNA levels in (A) MV4-11 and (B) MOLM13 cells were analyzed by quantitative real-time PCR at 0, 3, or 6 days after SYC-522 treatment. HOXA9 and MEIS1 expression were not detectable in (C) NB4 and (D) HL-60 cells. Only CCND1 and BCL2L1 mRNA expression were analyzed in these two cell lines. The mRNA expression was normalized to the level of 18S. Values represent the fold change in mRNA levels in treated vs. control cells. Bars represent the mean ±SEM of 3 independent experiments. *: p<0.05, day 0 vs. day 3 or 6, unpaired t-test.
Figure 3.
SYC-522 inhibited the colony formation ability of primary MLL-rearranged leukemia cells.
Bar charts show the average percent change in CFU numbers in (A) three independent primary MLL-rearranged AML samples, (B) three independent primary MLL-rearranged ALL samples, and (C) three independent normal bone marrow (NBM) samples, after SYC-522 treatment. CFUs were counted after 14 days and the values from duplicate dishes were averaged and expressed as percent of the untreated control. Values represent the mean ±SEM of 3 independent primary samples *: p<0.05, untreated vs. SYC-522 treated, One-way ANOVA followed by Tukey's post tests.
Figure 4.
SYC-522 promoted the differentiation of MLL-rearranged leukemia cell lines and primary MLL-rearranged AML cells.
(A) MV4-11 cells were treated with 3 µM SYC-522 and MOLM13 cells were treated with 10 µM SYC-522 for up to 18 days. Cells were analyzed by flow cytometry for CD14 expression. MFI (mean fluorescence intensity) ratio = MFI (SYC-522 treated)/MFI (untreated), n = 3 independent experiments, *p<0.05, unpaired t-test; (B) Primary MLL-rearranged AML cells were harvested after clonogenic assays and stained with Wright Giemsa (original magnification 40X).
Figure 5.
SYC-522 sensitized MLL-rearranged cells to mitoxantrone-induced apoptosis.
(A) MV4-11 cells were treated with 3 µM SYC-522 and (B–D) other cell lines were treated with 10 µM SYC-522 for 0, 3, or 6 days. Following pretreatment, mitoxantrone (10 nM or 100 nM) was added to cells for 24 h incubation before the measurement of cell apoptosis by Annexin V staining. Bars represent the mean ±SEM of 3 independent experiments. *: p<0.05, chemotherapy vs. chemotherapy +SYC-522, unpaired t-test. (E–F) Two primary MLL-rearranged AML samples were cultured in methylcellulose and treated with SYC-522 (10 µM) and chemotherapy (etoposide 100 nM or 1 µM; or mitoxantrone 10 nM or 100 nM). After two weeks, the total numbers of colonies were counted for each sample, and the values from duplicate dishes were averaged.
Figure 6.
SYC-522 impaired DNA damage response and promoted mitoxantrone- induced apoptosis in MV4-11 cells.
MV4-11 cells were pretreated with SYC-522 (3 µM) for 3 or 6 days, followed by 100 nM mitoxantrone for 4 h. Then mitoxantrone was washed away and cells were incubated in fresh medium for 12 h. The cells were then analyzed by flow cytometry for the percent viable and repairing (γH2AX+/cPARP−; blue gate) and the percent undergoing apoptosis (cPARP+; red gate). A representative series of dot plots is shown. Bars represent the mean ±SEM for 3 independent experiments. *: p<0.05, chemotherapy vs. chemotherapy +SYC-522, unpaired t-test.