Identification of a novel E-box binding PI polyamide inhibiting MYC-driven cell-proliferation

Abstract

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and over expression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5’-CACGTGT-3’) sequence at gene promoters contributes to more than 4,000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding Pyrrole-Imidazole (PI) polyamide, Myc-5, that recognizes E-box consensus sequence. Bioinformatics analysis revealed that Myc-5 binding sequence appeared in 5′ MYC binding E-box sequences at the eIF4G1, CCND1 and CDK4 gene promoters. Furthermore Chromatin immunoprecipitation (ChIP) coupled with detection by qPCR demonstrated that Myc-5 has the ability to inhibit MYC-binding at the target gene promoters and thus cause down-regulation at m-RNA level and protein expression of its target gene in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and K562 (human chronic myelogenous leukaemia) cell lines. Single intravenous injection of Myc-5 at 7.5 mg/kg dose caused a significant tumor-growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of an E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibits MYC dependent tumor growth.

Introduction

c-MYC is a transcription factor that possesses an exclusive and extensive set of biological actions that underlie its role as a salient oncogene and therefore could be the key to anticancer drug development. The c-MYC proto-oncogene belongs to the family of MYC genes that includes B-MYC, L-MYC, N-MYC, and S-MYC ⁽¹⁾. Among them c-MYC (here after referred as MYC) is found in almost all proliferating cells and expression of N- and L-MYC is more constrained to specific cell types ⁽²⁾. MYC is a basics-helix–loop–helix leucine zipper (bHLH-LZ) transcription factor that binds DNA in a sequence-specific manner ⁽³⁾ and activates the transcription of genes whose products are involved in crucial aspects of cancer biology such as cell proliferation, cell growth, apoptosis and differentiation ⁽⁴⁾. The biological activities of MYC depends on its ability to heterodimerize with its protein partner, MAX, to bind the Enhancer Box (E-box) sequence and stimulate transcription of a number of its downstream genes ^(4;5). The MAX:MAX homodimer or MAD:MAX heterodimer is supposed to antagonize the functions of MYC by binding to the same core E-box sequences ⁽⁶⁾.

MYC targets about 10–15% of all cellular promoters in human cells, which is higher than any other typical transcription factor ⁽⁷⁾. Target genes of MYC identified in mammalian cells, include genes involved in cell cycle, ribosomal biogenesis, protein synthesis and mitochondrial function ^(4;5;8). Among these direct target genes of MYC the one with E-box binding sites include ODC, ECA39, eIF4E, CDC25, CAD, CDK4, eIF4G1 and CCND1 genes ^(9;10). MYC has been known to play crucial role in malignant transformation ⁽¹¹⁾. Several attempts have been made using small molecule inhibitors to inhibit MYC binding at gene promoters ^(11–16). Pyrrole-imidazole (PI) polyamides are a class of sequence-specific DNA-binding small molecules that have been shown to be effective inhibitors of transcription factors by disrupting essential protein-DNA interactions ^(17–21). Our group and others have designed PI polyamides to specifically target critical regulatory proteins, including MMP-9, TGF-β1, VEGF, HIF1-α, AR, EGFR, PSA, and LOX-1 ^(17;22–25). Recently we have designed PI polyamides recognizing E-box, Myc-6 which significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo showing potential of PI polyamide in cancer therapy ⁽²⁶⁾.

In the present study, we developed an E-box binding PI polyamide, Myc-5 (target sequence; 5′-WCWCGWGW-3; where W= A or T) to inhibit MYC target genes. Myc-5 showed inhibition of MYC-driven cell-growth by down regulation of a subgroup of MYC downstream genes, including eIF4GI and CCND1 at early stage of transcription. In animal tumor model studies, Myc-5 inhibits tumor growth by inhibiting cell proliferation and inducing apoptosis in tumor tissue. Collectively, our results establish a transcriptional regulation at early stage of MYC regulatory proteins by using PI polyamides and provide a novel antitumor agent targeting MYC function.

Materials and Methods

Cell culture

The human Burkitt's lymphoma model cell line, P493.6, carrying Tet-repressible c-MYC system and chronic myeloid leukemia cell line K562, were used in this study. To suppress MYC expression P493.6 cells were treated with 0.1 µg/ml tetracycline for three days before treatment with Myc-5. P493.6 cell line were kindly provided by C. Grandori (FHRC, Seattle, Washington, USA) and K562 cells were obtained from the RIKEN cell bank (Ibaraki, Japan).

Synthesis of PI polyamides targeting the E-box consensus sequence

Myc-5 was designed to target the E-Box consensus sequence and mismatched PI polyamide was designed to target by exchanging CG dinucleotide with GC in the center of the E-box (Figs. 1A and 1C). Fluorescein isothiocyanate (FITC)-conjugated Myc-5 was also synthesized for nuclear localization experiments (Fig. 1B). All of the PI polyamides were synthesized according to previously established methods ⁽²²⁾.

Figure 1. Designed PI polyamide structure and binding site.

Structures and binding sites for synthesized PI polyamides A, Myc-5; B, FITC labeled Myc-5 C, Mismatch PI polyamide. Base sequence specificity depends on side-by-side pairing of Pyrrole and Imidazole amino acids in the minor groove of DNA. Black and white circles, Im and Py rings, respectively; curved line, hairpin junction,; β is β-alanine; Dp is diaminomethylene propylamide and W=A or T.

Electrophoretic mobility shift assay (EMSA) and Surface Plasmon Resonance (SPR)

FITC-labeled match 46 base pair hairpin oligonucleotide corresponding to eIF4G1 gene promoters having E-Box consensus binding sites and mismatch promoter were synthesized (Table S1) for EMSA. Results were visualized by luminescent image analyzer LAS3000 (Fuji film, Japan). The kinetic measurements of the polyamides binding curves to the biotin-labeled double-stranded DNA (having E-box consensus sequence) and data processing were performed on a Biacore 2000 system as described previously ⁽²⁷⁾.

Quantitative real-time PCR (qPCR)

Total RNA was extracted and digested with DNaseI using the RNeasy kit according to manufacturer’s protocol (QIAGEN). The primer sequences are listed in the Table S2. Relative gene expression was determined by normalizing the gene expression of each target gene to GAPDH.

qChIP (conventional ChIP followed by real-time PCR)

The status of polyamide binding at the target promoter region (Table S3) was detected using a ChIP assay kit (Upstate Biotechnology) following manufacturer protocol. ChIP DNA was further analyzed by qPCR using primers encompassing the regions of interest on the eIF4G1, CCND1 and CDK4 promoter.

Tumorigenicity studies in SCID mice

Seven weeks old SCID mice were housed under specific pathogen-free conditions. Experiments were approved by the committee for laboratory animal welfare and ethics of Nihon University School of Medicine, Tokyo, Japan. The effect of Myc-5 on the xenograft model was examined as follows: 1×10⁷ P493.6 cells were inoculated subcutaneously (s.c.) in the flank of the mouse. The mice were divided into three groups: a control group of (PBS i.v.; n=8), a Myc-5–treated group of (7.5 mg/kg i.v., single dose; n=8), and a doxycycline treated group: 0.01% of doxycycline in regular drinking water (n=5). Doxycycline-induces repression of P493.6 xenograft tumor growth in MYC-dependent manner. The treatment was started on day seven after cell inoculation and mice were sacrificed after 30 days of treatment.

In vivo nuclear localization

For in vivo nuclear localization analysis by fluorescence microscopy, tumor-bearing mice were injected with FITC-labeled Myc-5 (0.15 mg) into the lateral tail vein of the animals. Tumor tissues, along with adjacent normal tissues were collected five days after the injection for analysis using propidium iodide as a nuclear dye to identify nucleated cells.

Statistical analysis

Results were shown as mean SD. Each experiment was performed independently thrice. The level of significance (P values ** <0.05 and *** <0.001) was determined using the Student’s t test.

Additional supplementary material and method for cell viability assay, detection of nuclear localization by confocal microscopy, histopathology, western blot analysis, microarray analyses and references can be found in Doc S1 and Doc S2.

Results

Identification of MYC target genes that harbor Myc-5 consensus sequence

E-boxes are present in many genes, but not all are known to be direct transcriptional targets of MYC. Based on compilations by Fernandez et al. ⁽²⁸⁾, we found that 105 genes harbors the Myc-5 binding site (WCWCGWGW) in their E-box consensus sequence (Table S4). These genes can be classified into a wide range of functional classes. Here we focused on genes related to translation and proliferation. Among the top ten MYC-regulated genes, proliferation genes (CDK4, CCND1) and translation (eIF4G1) gene. Selected genes have been well characterized by previous researchers and are also well known direct targets of MYC ^(10;29;30).

DNA-Binding affinity and specificity of designed Myc-5 PI polyamide

EMSA and SPR assay are used to determine the binding affinity and specificity of PI polyamide to its target DNA. EMSA results demonstrated that a clear mobility band shift was (Fig.2A and 2B) detected when Myc-5 was incubated with eIF4G1 gene promoter oligonucleotide, whereas no shift was detected for the mismatched PI polyamide (Fig. 2A, lane 4) or eIF4G1 mismatch gene promoter (Fig. 2A, lane 5) in which the core recognition sequence, CACGTG, was replaced by CAGCTG. Increasing concentrations of the Myc-5, but not of the mismatch PI polyamide, bind to the eIF4G1 promoter oligo (Fig. 2B, lanes 2–5), suggesting that Myc-5 can specifically bind to sequences of their target gene-promoters. To further confirm binding of the E-box to the target gene promoter, we did a biosensor-SPR assay to explore the interaction of Myc-5 with biotinylated hairpin duplexes having WCWCGWGW sequences (Fig. 2C). The kinetic profile of Myc-5 revealed a relatively fast “on-rate” and slow “off-rate” in binding with a K_D of 4.81±2.79 × 10⁻⁸ M. The mismatched PI polyamide showed slow “on rate” and fast “off rate” with K_D was calculated at 5.1±0.54×10⁻⁵. In summary, these results confirm that the presence of the E-box in the DNA sequence has a pronounced influence on the binding of Myc-5 and yields a 654-times-higher affinity than the mismatched PI polyamide (Figs. 2D, 2E and Table S5).

Figure 2. Myc-5 binding at the target gene promoters.

A, EMSA of eIF4G1 gene match and mismatch promoter and with Myc-5 and mismatched PI polyamide. B, EMSA of eIF4G1 gene promoter with Myc-5 and mismatched PI polyamide. FITC-labeled hairpin oligonucleotide was incubated at 37°C for 60 min in Myc-5 or mismatched PI polyamide. C, Typical SPR sensograms for the interaction of PI polyamides with the hairpin duplex with 5’-biotin labeled and immobilized E-box (CACGTG) sequences. D–E, Remarkable differences in binding kinetics are observed: fast on/off kinetics for Myc-5 (D) and slower kinetics for the mismatch PI polyamide (E).

Myc-5 inhibited cell proliferation and localized into nucleus in P493.6 and K562 cell lines

P493.6 and K562 cells were incubated with different concentration (1–10 µM) of Myc-5 and mismatch PI polyamide and viability was determined at 24, 48, and 72 hours after treatment, respectively. As shown in Figs. S1A and S1B, cell viability was significantly reduced (P<0.001 versus control) in both cell lines treated with Myc-5 in a time- and concentration-dependent manner. Nuclear localization of Myc-5 was determined by FITC conjugated Myc-5 using laser confocal fluorescence microscopy. Green fluorescence indicates the presence of Myc-5, while red fluorescence depicts the cell nuclei, indicating that Myc-5 localizes into nucleus within two hours (Figs. S2A, S2C and S2D). On the other hand cells incubated with FITC solution (control) at same concentration do not localize into nucleus (Fig. S2B) in both cell lines.

Myc-5 attenuates MYC binding at the gene promoter causes down-regulation of MYC target genes

Myc-5 inhibited target gene expression at protein and m-RNA levels (Fig. 3A & 3B). Cells treated with Myc-5 at 10µM concentration for 72 hours caused statistically significant suppression of eIF4G1 mRNA as compared with control or mismatched PI polyamide treated cells in both systems. The CCND1 m-RNA was unaffected in the all treated and untreated groups of P493.6 cells; however, its expression was significantly (P < 0.001) down-regulated in the K562 cell line compared to control and mismatch PI polyamide treated group. The mRNA expression correlated well with protein expression in the Myc-5 administration using both cell systems (Fig. 3C &D). To investigate whether target genes expression are directly regulated by Myc-5, we did ChIP assays of E-box and the Exonic region (Figs. 4A, 4B and Fig S3A). The Exonic region is taken as arbitrary negative control region as during the analysis. ChIP analysis revealed that compared to control Myc-5 and tetracycline group significantly inhibits binding of MYC to E-Box region in P493.6 cell (Fig. 4C and Fig S3B) at the eIF4G1 and CDK4 gene promoters. In contrast E-box and control region (Exon) of CCND1 gene promoter showed restrained enrichment in all treated groups (Fig. 4D). Similarly, MYC was specifically enriched near the E-box site at the eIF4G1, CCND1 and CDK4 gene promoters but not Myc-5 treated group in K562 cells (Figs. 4E, 4F and Fig S3C). Moreover our microarray analysis results showed (Supplementary Figs. S4A and S4B) that differentially expressed genes in K562 cells (29 up-regulated and 21 down-regulated), in P943.6 cells (20 genes up-regulated and 20 down-regulated) were identified by unpaired two-class Significance Analysis of Microarray (SAM) analysis with 0% False Discovery Rate (FDR). Among top 50 modulated genes, five genes were carrying Myc-5 consensus sequence in their promoter as identified from Supplementary Table S4.

Figure 3. Myc-5 down-regulates m-RNA expression and protein expression of the target genes.

A-B, Gene expression of target gens were detected by quantitative real time PCR after 72 hour of control and treated with Myc-5 (1,5 and 10 µM conc.) and mismatch PI polyamide (MM) at 10 µM conc. and normalized with GAPDH in (A) P493.6 and (B) K562 cells. The data in A and B are shown as mean value with error bars representing ± SD. Statistical significance was calculated by Student's t test. **, p<0.05, **, p<0.001 when compared to control. C-D, P493.6 and K562 cells were treated with Myc-5 (1, 5 and 10 µM) and mismatch PI polyamide (10 µM) for 72 hours. Western blotting was performed as described in supplementary material method section. β-actin was used at loading control. The Relative band intensities in P493.6 and K562 cells was determined by dividing the intensity of the band by β-actin followed by normalization to the control.

Figure 4. In vivo binding of Myc-5 to the E-box at its target gene promoter.

A-B, Schematic depiction of the Myc-5 target gene promoter with MYC binding site (underline) indicated. C–D, ChIP assay of Myc-5 target genes in the P493.6 and E–F, K562 cell system. Labeled regions (E-box and Exon) of each gene were quantitatively amplified by real-time PCR. Data are representative of three independent experiments.

Myc-5 administration retards growth in animal tumor models

To investigate if the in vitro efficacy of Myc-5 can also be recapitulated in vivo, therapeutic animal studies were conducted using P493.6 subcutaneous xenografts. Mice were inoculated s.c. with P493.6 (high MYC expressing) cells. At one week after inoculating the cells, when the tumor volumes reached around 100 mm³, mice were split into three groups and treated with either saline, doxycycline or 7.5 mg/kg Myc-5 injected intravenously into the lateral tail vein animals at day 7 (single dose; Figure 5A). Myc-5 was formulated based on previous xenograft studies with PI polyamide ^(21;31;32). Growth curve data indicated that Myc-5 (7.5 mg/kg) and doxycycline treated groups had a significantly smaller in tumor volume during the growth phase (P<0.001 versus control; Fig. 5B) by the end of study. Representative image of each group mice is shown in Fig 5B (inset). All mice with Myc-5 treatment continued to gain weight at an equal rate throughout the treatment period (Fig. 5D). The average tumor weight result further confirmed inhibition of tumor growth as Myc-5 and doxycycline treated groups were found to be significantly lower (P < 0.001 versus control; Fig 5E) at the termination of study.

Figure 5. Myc-5 blocks the growth of P493.6 xenografts.

A, Schematic diagram of the xenograft model illustrating timing of tumor implantation and treatment. Eight weeks old SCID mice were subcutaneously injected with P493.6 cells. B, Tumor-growth chart showing the effect of treatment in vivo. Myc-5 (7.5 mg/kg) and doxycycline significantly slowed tumor growth (P<0.001) at termination point in comparison to control group. Representative picture of each group mice (inset). C, FITC-labeled Myc-5 localizes to nucleus of P493.6 xenograft leaving normal tissue unaffected (separated by white line). N: Normal tissue, T: tumor tissue. D, Mean body-weight for each treatment group plotted as function of day after post-injection. E, Comparisons of excised tumor weights for three different treatment groups at the end of study are shown. The data in B, D and E are shown as the mean ± SD. Statistical significance was calculated by Student's t test. ***P < 0.001.

Myc-5 localizes into the tumor and causes decreased cell proliferation and induced apoptosis in P493.6 tumor xenografts

To evaluate in vivo nuclear localization single intravenous injection of FITC-conjugated Myc-5 was given to P493.6 cell-derived xenograft. Twenty-four hours after intravenous injection of FITC-conjugated Myc-5 to xenografts, animals were killed and tumor tissues were obtained. The tumor-derived tissues were found to display strong and characteristic nuclear staining (Fig. 5C). In contrast, adjacent normal tissues found to be devoid of nuclear fluorescence (Fig. 5C). Myc-5 was found throughout the tumor, demonstrating its capacity of enter the tumor through the vascular system. To assess the activity of Myc-5, tumors were harvested from all treatment groups and examined by histopathology. Microscopic analysis of H&E staining showed that Myc-5 and doxycycline treated tumors exhibited areas with necrosis, cellular debris and swollen cells with cytoplasmic vacuoles as compared with the vehicle-treated control (Fig. 6A; left). In order to investigate the mechanism underlying Myc-5-mediated tumor growth inhibition in P493.6 xenografts, immunohistochemical analyses were conducted for BrdU uptake, and TUNEL reaction assay. BrdU-positive nuclei were detected in a small number of cells in Myc-5 treated tumor as compared to control group (Fig 6A; middle). In contrast a large number of cells stained positively for TUNEL in Myc-5 or Doxycycline treated group (Fig. 6A; left) compare to control group. Quantitative data were consistent with expression pattern of BrdU and TUNEL staining assays (Fig. 6B). Quantification of BrdU positive cells in control group were significantly (P<0.001) reduced (~83% and ~76% reduction in Myc-5 and doxycycline respectively) compared to treated groups. TUNEL analysis showed that number of apoptotic cells was significantly higher (P<0.001; ~38% and ~30% higher in Myc-5 and doxycycline respectively) in treated groups. Overall we found that Myc-5 is well tolerated, inhibits tumor growth and induces apoptosis in P493.6 xenograft mouse models.

Figure 6. Histopathology of xenografts in nude mice and illustration of potential mechanism by Myc-5.

A, Tissue sample were analyzed qualitatively for the morphological changes. Magnification of all pictures is ×10 (scale bar 200µm) and ×40 magnification (scale bar 50µm). B–C, Quantitative data of immunohistochemical analysis of BrdU and TUNEL positive staining in each group. The data in B and C are shown as the mean ± SD of three tumor samples from an individual mouse in each group.. Statistical significance was calculated by Student's t test. ***P < 0.001. D, Schematic diagram of the mechanism by which PI polyamide inhibits MYC/MAX interaction to E Box. (I), MYC/MAX dimer binds to E-box and activates MYC target gene expression. (II), Myc-5 occupied the E-box by binding, thereby inhibiting the MYC/MAX interaction to E-box further causing suppression of target gene expression.

Discussion

In this study we synthesized two β-alanine-linked polyamides of different lengths, Myc-5 (AcPyPyPyImβImγPyβPyImβImβDp), where Py is pyrrole, Im is imidazole, Ac is acetyl, β is β-alanine, Dp is dimethylaminopropylamine and γ is γ-turn to target the 8-bp site of 5′-WCWCGWGW-3′ and mismatched polyamide (AcPyβImPyPyImγPyPyImPyβImβDp) to target the 8-bp site 5′-WCWGCWGW-3′ flipping dinucleotides of CG to GC at the central portion (Figs. 1A, 1B). A search for Myc-5-binding sites from the published database ⁽²⁸⁾ revealed that the Myc-5 consensus sequence was flanking to a E-box of genes involved in apoptosis, cell cycle, nucleolar function, ribosomal proteins and translation initiation factors. Among them, we focused on the eIF4G1, CCND1 and CDK4 genes because they carry a Myc-5 consensus sequence including a MYC binding site (E-box) in the promoter region (Fig S3D) and their transcription are modulated by MYC ^(9;10;30). Using EMSA and Biacore analyses, our results demonstrated that Myc-5 binds specifically and with high affinity (654 times; Table S5) to the eIF4G1, CCND1 and CDK4 gene promoter at E-box region (Fig. 2). The effective concentration of Myc-5 in the EMSA was higher than the concentration for biological effect, which is consistent with results obtained for other compounds ⁽³³⁾; possibly, PI polyamide accumulates in cells to effectively reach the intracellular and intra-nuclear levels ⁽³⁴⁾. The higher binding affinity of Myc-5 in comparison to mismatch PI polyamide might be explained as the targeted PI polyamide has more aliphatic β-alanine unit giving it more flexibility and optimizes the positioning of the imidazole amino acids on binding to its targeted sequence ⁽³⁵⁾. In vivo binding of Myc-5 to their target gene-promoter was confirmed by ChIP assay. Results of ChIP demonstrated that MYC transcription factor binds E-box in control group whereas in treated group Myc-5 inhibits MYC binding on its target gene promoters. However in the P493.6 cell lines the CCND1 gene-promoter showed only background signal that was obtained at the E-box region as well as in the control (Exon) regions (Fig. 4D). These results were consistent with previous studies reports of the absence of this regulator in the B cells line ⁽³⁶⁾.

Myc-5 is used at the concentration of 10 µM based on previous studies using PI polyamides ^(38–40). Myc-5 significantly reduced m-RNA and protein expression of MYC target genes at this given concentration. The correlation between the MYC binding and m-RNA expression of Myc-5 was established by real-time m-RNA expression analysis and western blot analysis. The eIF4G1 gene has a down-regulated expression and might be a direct target of MYC in both systems, which was also favored by EMSA and ChIP data. CCND1 m-RNA expression was down-regulated in the K562 cell line with Myc-5 treatment in a dose-dependent manner, which is known to exert positive growth effects and to be regulated by MYC ⁽³⁷⁾. The m-RNA expression of CDK4, a positive growth controller of MYC ⁽³⁰⁾, was unaffected by Myc-5 treated group as Myc-5 binding site (E Box#2) has no role in MYC dependent gene regulation (30). We analyzed the CDK4 promoter region for putative MYC binding sites. Our sequence analysis revealed the presence of four E-box sequences, which could be recognized by MYC (Figure S2). Among them only E-box#2 is a putative binding site for Myc-5 as it contains Myc-5 consensus sequence. As shown in Figure S2, Myc-5 bound to the E-box#2 of the CDK4 promoter. CDK4 gene is established as direct target of MYC identified by serial analysis of gene expression with essential E boxes in their promoters ⁽³⁰⁾. The CDK4 promoter contains four highly conserved E-box elements where E box#3 and #4 were the most important for CDK4 promoter activity ⁽³⁰⁾. Effect of Myc-5 on m-RNA expression on the MYC gene and further its transcriptional target CDK4 gene by real time PCR in K562 and P493.6 cell lines. In K562 cell MYC m-RNA expression is unaffected by Myc-5 treatment and similar trend was observed in its target gene CDK4 m-RNA expression. On the other hand in P493-6 B lymphoma cells cultured with (MYC-off) or without (MYC-on) tetracycline. P493.6 cells, MYC m-RNA expression is induced by Myc-5 treatment with respect to control group. P493.6 B lymphoma cells are MYC over expressing cell line which indicates P493.6 cell lines may have some mutations on its promoter or regulator regions. MYC promoter or regulatory region in do not have any Myc-5 binding consensus sequence. May be due to some specific mutation in the MYC promoter or regulatory regions of have generated a novel Myc-5 binding site at the MYC promoter of P493.6 cells. This novel Myc-5 binding site in the promoter is acting as a transcriptional activator of the MYC gene and helping it to further over express. Furthermore, Myc-5 has very high affinity for its consensus sequence and may be act as a non-competitive inhibitor of MYC protein. Therefore, over expression MYC protein in P493.6 cells does not have an effect on MYC downstream genes with active Myc-5 binding site such a eIF4G1.

Taken together, our data are compatible with a mechanism that involves the recruitment of Myc-5 at E-Box sites within the promoter of the MYC target gene thereby inhibiting MYC binding at particular site and inhibits target gene function instead blocking MYC. Myc-5 down-regulates its target gene transcription supporting the notation that polyamides bind to DNA with affinity and sequence-specificity comparable to DNA-binding proteins and gene expression can be regulated by competitive displacement of transcription factor from DNA target sequences ⁽⁴¹⁾, where Myc-5 consensus sequences are not contained.

Myc-5 exerts its therapeutic role in tumor maintenance via selective effects on the translation of specific downstream genes. In our in vivo study we have used 7.5 mg/kg on the basis of prior pharmacokinetic profile and previous analytical studies ^(38;42). We identified the strong in vivo nuclear localization of Myc-5 and also inhibitory effect on the tumor growth in P493.6 mouse model. One possible reason of this activity can be explained by previous reports showing that changes in the levels or activity of eIF4F mediate the translational regulation of specific genes involved (as in P493.6 cell line eIF4G1 is completely suppressed) in survival and apoptosis ⁽⁴³⁾. In nasopharyngeal carcinoma, knockdown of eIF4G1 expression markedly inhibited cell-cycle progression, proliferation, and suppressed in vivo xenograft tumor growth ^(44;45), which might be applicable to our system as well.

In conclusion, the results reported here identify a novel PI polyamide, Myc-5 as a lead compound targeting against the E-box and thereby modulating expression of some genes. Our approach can be an efficient tool to identify the sequence-specific compound that targets the E-box and specifically modulates MYC downstream genes and interfere with their pathway. Myc-5 competes with the MYC: MAX heterodimer and inhibit trans-regulation of the target gene promoter (Fig. 6D) at the E-Box. However, the extension of this approach could result in the identification of more potent inhibitory PI polyamides in multiple pathways and reveal a new E-box-regulated target or combination of targets for a distinct MYC function, which may have formidable therapeutic opportunities for the future.