Abstract Many cancers harbor reciprocal chromosomal translocations that can lead to the activation of proto-oncogenes, inactivation of tumor-suppressor genes and/or generation of oncogenic chimera. In Non-Hodgkin Burkitt's lymphoma (BL) there is a characteristic translocation of the c-MYC gene on chromosome 8q24 to one of three immunoglobulin genes on chromosomes 14, 2, or 22. The resulting product leads to c-MYC upregulation. ∼90% of c-MYC gene transcription is initiated by the P1 and P2 promoters, just upstream of which lies the guanine-rich NHE III1 control element. This is a very dynamic stretch of DNA, capable of forming at least three different DNA topologies: single stranded, double stranded and the G-quadruplex (G4) DNA. The G4 is a transcriptional silencer, and thus an attractive target for anti-cancer therapeutics. Using two BL cell lines (RAJI and CA46) we directly demonstrate the formation of a G4 in the c-MYC promoter. Using a small molecule compound, we clearly show specific activity dependent on the existence and regulation of this structure. The BL reciprocal translocation t(8;14) maintains G4-mediated control of c-MYC on both chromosomes in RAJI, but only on the non-translocated (NT) chromosome in CA46. The translocated (T) chromosome's rate of transcription is 1000-fold greater than the NT, making the T the major allele, and the NT the minor allele. Thus, RAJI harbors G4-mediated control of both alleles and is expected to be more sensitive to G4-interactive compounds than CA46, which lost G4 control on the major allele. Indeed, GQC-05 (NSC338258), previously demonstrated to bind and stabilize the c-MYC G4, had greater cytotoxicity in the RAJI cell line. This compound demonstrated a rapid and time-dependent downregulation of c-MYC mRNA in RAJI, but no overt c-MYC effect in CA46. We were able to distinguish, using primers specific to exons 1 or 2, between the NT and T mRNA products, respectively. Remarkably, GQC-05 rapidly and significantly downregulated c-MYC mRNA from the NT allele in CA46 cells, where the G4 maintains transcriptional control. As a negative control, we examined the effects of GQC-05 on both exons in RAJI cells; no differential effect was observed. Doxorubicin is a non-G4 interactive compound that also decreases c-MYC expression in CA46 cells, but does not show this ‘exon-specific’ effect. Chromatin immunoprecipitation confirmed GQC-05 changes protein binding to the G4-region of the c-MYC promoter, with decreased transcription factor, and altered G4-regulatory factor, binding. Our work elucidates the primary mechanism of action for GQC-05 as stabilization of the c-MYC G4, leading to transcriptional downregulation. More importantly, we unambiguously demonstrate the formation and regulation of the c-MYC G4 in vitro and for the first time are able to evaluate c-MYC G4-targeted compounds in a whole cell system with this newly described CA46 model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4422. doi:10.1158/1538-7445.AM2011-4422