Abstract 3811: G-quadruplex-forming genomic sequences homologous to Pu27 interact with c-Myc promoter and regulate c-Myc transcription

Introduction: The transcription factor c-Myc regulates a large array of genes, plays an important role in cell growth/differentiation, cell cycle progression and apoptosis hence is tightly regulated both at the transcriptional and protein level. The NHEIII1 upstream of P1 promoter regulates up to 80% of c-Myc transcription, and comprises a C/G rich region able to form G-quadruplex structure (Pu27). G-quadruplex forming motifs seem to be abundant in the genome, located in the promoter region of oncogenes. We identify 13 new potential G-quadruplex forming motifs with more than 90% identity with Pu27, located in different chromosomes of the human genome and none near gene promoter regions. Methods: BLAT search was used to identify the homologous sequences of Pu27. The G-quadruplex formation was evaluated by Circular Dichroism Spectrometry (CD), the specific interaction of each of the homologous sequences with the double strand DNA containing NHEIII1/Pu27 was documented by electrophoretic mobility shift assay (EMSA). We investigate 3 members of the Pu27 oligonucleotides family: Pu5- (42nu), Pu9- (20nu) and Pu27 (27nu) in: 1) a dose response (1, 3, 5 or 10µM)-time course (24, 48, 72 , 96 hrs) study on cell growth inhibition in 4 leukemia cell lines measured by MTT. 2) the effect on c-Myc expression in U937 measured by QRT-PCR, Flow-cytometry and Western Blot. 3) global gene expression evaluated in a gene array study in U937 exposed for 24h, 48h and 72h. Results: Thirteen oligonucleotide sequences almost identical to Pu27 were identified spread-out in 12 different chromosomes. Except for Pu27 (c-Myc promoter) none of the sequences were within a promoter region. All Pu27 homologous sequences contain the 5 runs of 3 to 4 Guanines, form G-quadruplex, and specifically bind to the dsDNA containing Pu27 sequence. The evaluation of a dose response/time course for Pu27 (Chr8), Pu5- (Chr5) and Pu9- (Chr9) on growth inhibition of U937, K562, HL-60 and Raji show a difference in sensitivity of the cell lines to the oligonucleotide sequences (Raji (more sensitive than)>U937>K562>HL-60), and a difference in efficiency of the oligonucleotide sequences (Pu27>Pu5->Pu9-). Gene and protein expression of c-Myc were similarly down regulated by the 3 sequences in U937 exposed to Pu27, Pu5- or Pu9- for 3 days. The analysis of the global gene expression reveal an early effect (24h) of Pu27 followed by Pu5- (48h), by 72h exposure all three Pu oligonucleotides will have affected genes involved in cell cycle progression/regulation, DNA damage repair and histone modification . Conclusion: We identify the presence of numerous sequences homologous to Pu27 located in different chromosomes sharing Pu27 structure and function. The addition of exogenous Pu27, Pu5- or Pu9- down regulate c-Myc expression and inhibit leukemia cells growth making this G-quadruplex structure a key regulator in NHEIII1 and a potential target for cancer therapy. Citation Format: Francine Rezzoug, Shelia D. Thomas, Eric C. Rouchka, Donald M. Miller. G-quadruplex-forming genomic sequences homologous to Pu27 interact with c-Myc promoter and regulate c-Myc transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3811. doi:10.1158/1538-7445.AM2014-3811