Inactivation of Receptor Tyrosine Kinases Reverts Aberrant DNA Methylation in Acute Myeloid Leukemia

Abstract

Purpose: Receptor tyrosine kinases (RTKs) are frequently deregulated in leukemia, yet the biological consequences of this deregulation remain elusive. The mechanisms underlying aberrant methylation, a hallmark of leukemia, are not fully understood. Here we investigated the role of RTKs in methylation abnormalities and characterized the hypomethylating activities of RTK inhibitors.Experimental Design: Whether and how RTKs regulate expression of DNA methyltransferases (DNMTs), tumor suppressor genes (TSGs) as well as global and gene-specific DNA methylation were examined. The pharmacologic activities and mechanisms of actions of RTK inhibitors in vitro, ex vivo, in mice, and in nilotinib-treated leukemia patients were determined.Results: Upregulation of RTKs paralleled DNMT overexpression in leukemia cell lines and patient blasts. Knockdown of RTKs disrupted, whereas enforced expression increased DNMT expression and DNA methylation. Treatment with the RTK inhibitor, nilotinib, resulted in a reduction of Sp1-dependent DNMT1 expression, the diminution of global DNA methylation, and the upregulation of the p15INK4B gene through promoter hypomethylation in AML cell lines and patient blasts. This led to disruption of AML cell clonogenicity and promotion of cellular apoptosis without obvious changes in cell cycle. Importantly, nilotinib administration in mice and human patients with AML impaired expression of DNMTs followed by DNA hypomethylation, TSG re-expression, and leukemia regression.Conclusions: Our findings demonstrate RTKs as novel regulators of DNMT-dependent DNA methylation and define DNA methylation status in AML cells as a pharmacodynamic marker for their response to RTK-based therapy, providing new therapeutic avenues for RTK inhibitors in overcoming epigenetic abnormalities in leukemia. Clin Cancer Res; 23(20); 6254-66. ©2017 AACR.

©2017 American Association for Cancer Research.

Figures

Figure 1

KIT and FLT3 upregulate DNMT expression in AML cells. A and B, The analysis of the GEO dataset GSE12417 series, platform GPL570 and GPL96, AML, n = 242 (A) and qPCR results of 9 leukemia cell lines (B) showing the correlation between FLT3 or KIT and DNMT gene expression, which was assessed by Spearman correlation. C, Kasumi-1 or MV4-11 cells were transfected for 48 h with FLT3 or KIT siRNA (si-) or scrambled control and the expression of targeted genes was detected by Western blot analysis. D, MV4-11 or Kasumi-1 cells were transfected for 48 h with FLT3 or KIT siRNA, respectively, or scrambled control. The genomic DNA was subjected to dotblot analysis. Graphs are the quantification of dot intensity. E, Quantitative analysis for colony-forming assays of Kasumi-1 or MV4-11 cells transfected with FLT3 or KIT siRNA or scrambled control. F, K562 or KU812 cells were transfected for 48 h with BCR siRNA and the cell lysates were subjected to Western blot analysis. G, Kasumi-1 or MV4-11 cells were transfected for 48 h with mTOR siRNA or scrambled control. The cell lysates were subjected to Western blot analysis. H, Quantitative analysis for colony-forming assays of K562 and KU812 cells transfected with BCR siRNA (left) or MV4-11 and Kasumi-1 cells with mTOR siRNA (right). In C, D, F and G, data represent 3 independent experiments; In E and H, the assays were performed in triplicate; In D, E and H, data are shown as mean values ± S.D; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Sp1 activity accounts for the suppression of DNMT1 expression in AML cell lines treated with nilotinib. A-C, Kasumi-1 or MV4-11 cells were treated for 24 h with the indicated doses of nilotinib and subjected to qPCR or Western blot analysis to detect the expression levels of DNMT1, DNMT3a, DNMT3b (A,B) and Sp1 (C). D and E, EMSA assays were used to detect Sp1 binding to the DNMT1 promoter in Kasumi-1 (D) or MV4-11 (E) cells treated for 24 h with nilotinib. In D a vertical line was inserted to indicate a repositioned gel lane. F and G, Reporter gene assays were used to determine DNMT1 gene promoter activity in 293T cells transfected with the DNMT1 promoter-luciferase plasmids together with the Sp1 expression vectors (F) or treated for 24 h with nilotinib (G). H, Western blot analysis of Kasumi-1 and MV4-11 cells transfected for 48 h with Sp1 siRNA (si-) or scramble control. I, Kasumi-1 and MV4-11 cells were transfected with Sp1 expression or control vectors for 12 h and then treated with 10 μM nilotinib for another 24 h. Gene expression was assessed by Western blot analysis. Data represent 3 independent experiments.

Figure 3

Nilotinib induces global and gene promoter DNA hypomethylation. A, Dotblot analysis using anti-5mC to evaluate changes in global DNA methylation in Kasumi-1 or MV4-11 cells treated for 24 h with nilotinib. B and C, Kasumi-1 or MV4-11 cells were transfected for 48 h with DNMT1 (B) or Sp1 siRNA (C) or scrambled control. The protein expression levels were assessed by Western blot (upper) and the levels of global DNA methylation were determined by dotblot analysis (lower). D, Kasumi-1 cells were transfected for 12 h with DNMT1 or Sp1 siRNA or scrambled control and then treated with 10 μM nilotinib for another 24 h. Global DNA methylation was determined by dotblot analysis. E, qPCR analysis of p15INK4B expression in Kasumi-1 or MV4-11 cells treated for 48 h with 10 μM nilotinib. Values are shown as a fold change of gene expression normalized to 18S RNA and compared to vehicle. F, Upper: diagram of the p15INK4B promoter indicating the location of CpG nucleotides; lower: bisulfite sequencing analysis of changes in p15INK4B promoter methylation (transcription start site +147 to +221) in Kasumi-1 or MV4-11 cells treated for 48 h with 10 μM nilotinib. Results of 10 clones are presented. Methylated CpG sites are shown as solid circles and open circles indicate non-methylated CpG sites. In A-D, the graphs show the quantification of dot intensity as mean values ± S.D. from 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001; si- = siRNA; LC = loading control.

Figure 4

Nilotinib suppresses growth and promotes apoptosis in AML leukemia cell lines. A-C, Kasumi-1 and MV4-11 cells were treated with nilotinib at concentrations of 0, 10 or 30 μM. (A) Colony-forming assays show the colony number as mean values ± S.D. from 3 independent experiments. (B) Flow cytometry was used for analysis of apoptosis and data are shown as a fold change in apoptotic cells compared to untreated control. (C) Western blot analysis was used to assess the levels of cleaved caspase forms. D, Kasumi-1 and MV4-11 cells were transfected for 12 h with DNMT1 siRNA (si-) or scrambled control and then treated with 10 μM nilotinib for another 24 h. Western blot detected cleaved caspase isoforms. E and F, Kasumi-1 and MV4-11 cells were treated with 5 nM velcade (Vel) or/and 3 μM nilotinib (Ni) and subjected to dotblot analysis (E) or colony assay (F). Data are shown as mean values ± S.D. from 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Nilotinib administration induces leukemia regression in mice. A, Approximately 0.1×106 C1498 cells were injected into C57BL/6 mice through the tail vein. When the white blood cell counts showed the illness, the leukemic mice were treated with vehicle or nilotinib for 3 weeks. External view (left) and quantification of spleen weight (right) from leukemia-bearing mice are shown. Data are presented as mean values ± S.D. B, Photographs (left) are representative external views of livers from leukemia-bearing mice and the graph (right) shows the quantification of liver weight. Data are shown as mean values ± S.D. C, Pictured are images of lung from leukemia-bearing mice. D, Representative images of H&E stained sections of lungs, livers and spleens from leukemia-bearing mice are shown (magnification × 200). E, Wright-Giemsa stained BM cells from leukemia-bearing mice are shown (magnification × 400). F, qPCR was used to determine the expression of DNMT1, DNMT3a, DNMT3b and Sp1 in BM cells from leukemia-bearing mice. G, The genomic DNA in BM cells from leukemia-bearing mice was isolated and subjected to dotblot analysis. H, qPCR was used to determine the expression of p15INK4B in BM cells from leukemia-bearing mice. Graph shows the quantification of dot intensities. Note, n = 3 mice/group; data are presented as mean values ± S.D; *P < 0.05, **P < 0.01.

Figure 6

Nilotinib suppresses DNMT expression, induces DNA hypomethylation and impairs AML patient cell expansion ex vivo and in vivo. A-G, AML patient (Pt) primary cells (n = 3) were treated for 24 h with 0, 10 or 30 μM nilotinib. (A) Cell lysates were subjected to Western blot analysis with the indicated antibodies. (B) The genomic DNA was subjected to dotblot analysis to assess changes in global DNA methylation. (C) qPCR analysis was used to determine p15INK4B gene expression. (D) Bisulfite sequencing was used to examine p15INK4B promoter DNA methylation. Methylated CpG sites are shown as solid circles and open circles indicate non-methylated CpG sites. A representative 10 clones are shown in the dot plot. (E) Colony-forming assays show cell proliferation. (F) Cellular apoptosis was determined by flow cytometry. (G) Western blot analysis was used to detect the activated forms of caspases. H-J, The PBMCs from AML patients (n = 14) receiving nilotinib therapy (day 0, day 7 or day 14) were subjected to qPCR for gene expression (H, J) or dotblot analysis for changes in global DNA methylation (I). In B, C, E, F, H, I and J, data are shown as mean values ± S.D; *P < 0.05, **P < 0.01.