Abstract 2607: G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies

Abstract

Abstract Introduction: Many cancer-related pro-oncogenes have quadruplex forming oligonucleotide sequences in their promoter regions. These genes include, amongst others, c-Myc, Bcl-2, K-ras, or HIF-1a which are individually or together over-expressed in the vast majority of neoplasms. For instance, c-Myc, a key regulator of cell cycle and proliferation, is commonly over-expressed in both hematological and solid cancers. The c-myc promoter region contains a guanine-rich sequence (PU27), capable of forming four-stranded quadruplex DNA structures, which negatively regulates c-myc transcription. Also, Bcl-2, is commonly over-expressed in a variety of hematological malignancies and the Bcl-2 quadruplex-forming sequence is located upstream of the P1 promoter of the Bcl-2 gene; it is also implicated in negative regulation of Bcl-2 transcription. Treatment of cancer cells over-expressing c-Myc and / or Bcl-2 with oligonucleotides encoding the c-Myc (PU27) and Bcl-2 (Bcl-2-GRO) guanine-rich sequences, located in their respective promoter regions, selectively inhibits c-Myc or Bcl-2 gene expressions, cancer cells proliferation and induces cell death. Methods: Four cancer cell lines obtained from ATCC were used in these studies (Raji, HL-60, U937, A549). Cell Titer-Glo© Assay was used for the cell proliferation experiments. Cell cycle analysis was determined in the HL-60 cell line using fixed doses of PU27 and Bcl-2-GRO and time dependent FACS analysis. Internalization studies were analyzed by FACS analysis using FAM labeled PU27 and DY647 labeled Bcl-2-GRO. In vivo tumor growth inhibition studies were done in females NOD/SCID mice (6 animals per group); 5x106 cells in a Matrigel mixture were implanted in the flank of the animals; tumors developed and treatments with PU27 and Bcl-2-GRO at different dose levels alone and in combination were performed while body weights were monitored during the course of the study. Results: Treating the Raji, HL-60, U937, or A549 cancer cell lines with PU27 or Bcl-2-GRO causes inhibition of proliferation with IC50 between 200 nM and 5mM after 144 hours depending on expression of the target pro-oncogene. When treating the same cell lines using a combination of these two sequences, a significant synergy is observed, leading to more than 100 fold increase in potency. The same synergistic effect was seen when PU27 was combined with paclitaxel, leading to picomolar IC50. Internalization of these oligonucleotides was very rapid and quantitative. Localization studies show that PU27 and Bcl-2-GRO are localized in the nucleus. Cell cycle results showed a significantly increase in G0-G1 cell cycle arrest for the combination. In vivo xenograft tumor growth inhibition studies confirmed the in vitro synergistic results with 60 to 80% tumor growth inhibition while treatments were well tolerated (no body weight loss). Citation Format: Gilles H. Tapolsky, Kara Sedoris, Shelia Thomas, Francine Rezzoug, Donald M. Miller. G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies. [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 2607. doi:10.1158/1538-7445.AM2014-2607