Abstract 1629: Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription
Authors: Felsenstein Kenneth M. , Simmons John K. , Gareiss Peter , Mock Beverly A. , Schneekloth John ‘Jay’ S. 
Published: May 2015

Abstract
The transcription factor c-myc, deregulated in ∼70% of all cancer cases, facilitates tumor initiation and promotion most frequently in colon, breast, lung, ovarian, and lymphoid malignancies, and acts as a universal amplifier of aberrant growth-related cell signaling. Due to its helix-loop helix topology, short protein half-life, and rapid replenishment within the cancer cell, c-myc was long considered to be ‘undruggable.’ Recently, much attention has focused on inhibiting c-myc either through indirect pathway modulation, or on the transcriptional level. 85-90% of c-myc gene expression is controlled by a G-quadruplex (G4) DNA structure found in the nuclease hypersensitive element III(1) region (NHEIII1) of its promoter, which, when stabilized with a chemical agent, would theoretically inhibit myc transcription by preventing normal polymerase function. In this study, we report the identification and biological characterization of a novel chemical class to directly bind the c-myc G4 DNA. The chemical motif discussed here was identified from a highly selective (hit rate of 0.16%, with few false positives) small molecule microarray screen of 20,000 compounds. A panel of the 12 most promising hits was chosen for biological and biochemical secondary validation in a variety of in vitro and cell based assays to confirm mechanism of action and functional effects in biological systems. Of note, numerous compounds from the initial panel of hits exhibited effects with a range of potency in comparison with untreated and non quadruplex forming nucleic acid controls, thereby validating the original screening method. For prioritization, hits were further evaluated for their capacity to preferentially induce myc-dependent cell death in a tetracycline controlled c-myc stable cell line. A top lead was identified, and structure-activity relationship was preliminarily evaluated with a small panel of analogues, leading to a final compound with three times greater potency. The chosen compound exhibited strong dose-dependent cell killing activity, in human multiple myeloma with high levels of c-myc, with an IC50 in the single digit micromolar range. Additionally, the compound reduced c-myc gene expression in a series of B cell neoplasms to near the limit of detection, both transcriptionally and translationally. The mechanism of action was confirmed in a Burkitt's Lymphoma model through a quadruplex promoter-specific comparative analysis of myc transcript copy numbers in the presence or absence of treatment. Continuing studies will focus on further structural optimization of the ligand, a thorough examination of its genome-wide effects, and tolerability in animal models.
Citation Format: Kenneth M. Felsenstein, John K. Simmons, Peter Gareiss, Beverly A. Mock, John ‘Jay’ S. Schneekloth. Identification and biological characterization of a novel class of small molecules to inhibit 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 1629. doi:10.1158/1538-7445.AM2014-1629