Specific Inhibition of DNMT3A/ISGF3γ Interaction Increases the Temozolomide Efficiency to Reduce Tumor Growth

Abstract

DNA methylation is a fundamental feature of genomes and is a candidate for pharmacological manipulation that might have important therapeutic advantage. Thus, DNA methyltransferases (DNMTs) appear to be ideal targets for drug intervention. By focusing on interactions existing between DNMT3A and DNMT3A-binding protein (D3A-BP), our work identifies the DNMT3A/ISGF3γ interaction such as a biomarker whose the presence level is associated with a poor survival prognosis and with a poor prognosis of response to the conventional chemotherapeutic treatment of glioblastoma multiforme (radiation plus temozolomide). Our data also demonstrates that the disruption of DNMT3A/ISGF3γ interactions increases the efficiency of chemotherapeutic treatment on established tumors in mice. Thus, our data opens a promising and innovative alternative to the development of specific DNMT inhibitors.

Keywords: DNA methylation; DNMT; DNMT inhibitor; GBM.; epigenetic; glioma.

Figure 1

A high level of DNMT3A/ISGF3γ interaction correlates with a poor level of sensitivity to temozolomide/irradiation-induced cell death. A. Graph illustrates the correlation existing between the overall survival (OS) of 31 GBM patients and the percentage of temozolomide/irradiation-induced cell death (TMZ/Irrad-induced cell death) of primary cultured tumor cells (PCTC) issue to the corresponding GBM. A circle represents a couple patient/PCTC issues from the considered patient. p and r values were obtained by performing a Pearson's test. B. Schematic representation of the temozolomide/irradiation-induced cell death. C. Graph illustrates the existing correlation between the percentage of temozolomide/irradiation-induced cell death and the number of DNMT3A/D3A-BP interaction of interest. The number of DNMT3A/D3A-BP interaction was estimated by P-LISA. A circle represents a PCTC. p and r values were obtained by performing a Pearson's test.

Figure 2

A high level of DNMT3A/ISGF3g interaction is a poor prognosis factor. Kaplan-Meier curves illustrate the difference of overall survival (OS) between patient with high (H) and low (L) levels of DNMT3A/ISGF3γ interaction. p value is obtained by performing a Cox Proportional Hazards Survival Regression test.

Figure 3

Specific disruption of DNMT3a/ISGF3γ interaction. A. Graph illustrates the binding intensities of GST-ISGF3γ for DNMT3A-derived peptides after quantification using ImageJ software as described in material and methods section and in figure S1. The binding regions were shown on the schematic representation of the DNMT3A protein. The sequence of DNMT3A-derived peptides used to challenge the DNMT3A /ISGF3γ interactions was here shown. B and C. Impact of peptides miming the DNMT3A/ISGF3γ binding regions on the DNMT3A/ISGF3γ interaction. Pictures and graphs are representatives of three independent pull-down experiments. I: input. Sequences of DNMT3A-derived peptides are shown in figure 3A. p values were obtained by performing a t test. D. Impact of peptides miming the DNMT3A/ISGF3γ binding regions on the DNMT3A/ISGF3γ interaction. Pictures and graphs are representatives of three independent P-LISA experiments. Red dots represent the DNMT3A/ISGF3γ interactions or close proximity. Graph illustrates three independent experiments. p values were obtained by performing a t test.

Figure 4

Specific effect of P1. A. Graph illustrates the fact that P1 did not affect the DNMT3A/AP2α, DNMT3A/GATA1 and DNΜΤ3Α/HDAC1 interactions. p values were obtained by performing a t test. B. Representation of the dose-schedule of treatments (left). Graph (right) illustrates the fact that P1 did not affect the global DNA methylation level. Global DNA methylation level was monitored by ELISA using Methylamp Global DNA methylation Quantification kit (Euromedex-Epigentek, France). p values were obtained by performing a t test.

Figure 5

Impact of P1 on cancer hallmarks/phenotypes. A. Schematic representation of the temozolomide/irradiation-induced cell death. Percentages of cell death were evaluated by using a Trypan Blue Stain 0.4%, and the Countess® Automated Cell Counter (Life Technology, France). p values were obtained by performing a t test. B. Doubling time (i.e. the period of time required for a quantity to double in size) was calculated by using the http://www.doubling-time.com/compute.php website and counting the proliferation of 10³ cells during 120 hours. Migration index was calculated by performing a scratch test. Invasion index was estimated by using cell invasion assay according to the manufacturer's instructions (Millipore, France). Graphs illustrate the average±SD of 3 independent experiments. p values were obtained by performing a t test. C. Score of Modulation of Cancer Hallmarks (SMoCH) was calculated by attributing -1 when the peptide/treatment enhanced a cancer hallmark, 0 when peptide/treatment did not modify a cancer hallmark and +1 when the peptide/treatment inhibited a cancer hallmark. By taking into consideration 4 cancer hallmarks/phenotypes, a worst anticancer treatment theoretically obtains SMoCH=-4, while an ideal anticancer treatment theoretically obtains SMoCH=+4.

Figure 6

Effect of a treatment associating the P1 peptide with TMZ in a swiss nude mice model of established tumors. A. Design of the experiment. Tumor establishment indicates that 2.10⁶ PCTC-GBM were injected to form a tumor of which the volume was equal to 100mm³±33.3. Then, mice were treated with indicated treatment. D: day, w: week, it: intra-tumoral, ip: Intraperitoneal. B. Graph illustrates the impact of the 4 considered treatments on tumor weight of established tumors. Open circles represent mice. Black circles represent the average±standard deviation obtained for each treatment. p values were obtained by performing a t test.

Figure 7

Impact of P1 treatment on MGMT methylation. Cells were treated with P1 as described in figure 4. qMSP were next performed to analyze the MGMT methylation status as described previously. p values were obtained by performing a t test.