ABSTRACT
The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Lactams/pharmacology , Polycyclic Compounds/pharmacology , Proto-Oncogene Proteins c-myc/genetics , Repressor Proteins/metabolism , Small Molecule Libraries/pharmacology , Transcription, Genetic/drug effects , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Line , Dimerization , Disease Models, Animal , Humans , Lactams/chemical synthesis , Lactams/therapeutic use , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasms/drug therapy , Polycyclic Compounds/chemical synthesis , Polycyclic Compounds/therapeutic use , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins c-myc/metabolism , Rats , Repressor Proteins/chemistry , Repressor Proteins/genetics , Small Molecule Libraries/therapeutic use , Ultraviolet RaysABSTRACT
We have evaluated the feasibility of using fusion genes to link transcriptional promoters of biomedical interest to reporter genes to screen for pharmacological or genetic regulatory effects. Using gene targeting, we generated two lines of embryonic stem (ES) cells expressing human alpha-fetoprotein (hAFP) under control of the endogenous promoter for the LDL receptor (LDLR). In one line, hAFP was introduced into the first intron after the translational start codon; in the other, hAFP was positioned in the 3'-untranslated region leaving the potential for expression of LDLR intact. In both lines, an internal ribosome entry site (IRES) was included to facilitate translation. Readily measurable levels of hAFP were found in the medium with both targeted ES cell lines, compared with undetectable levels with the starting cell line. The expectation that the level of hAFP would reflect the steady-state level of mRNA for the fusion transcript and secondarily transcriptional control of LDLR was confirmed by correlating hAFP levels with the abundance of LDLR and fusion transcripts. We also generated mice carrying the LDLR-hAFP fusion in the 3'-untranslated region and these mice produced detectable levels of hAFP in serum. Levels of hAFP in culture medium and in serum were increased by simvastatin, a drug known to up-regulate LDLR. These ES cell clones and mice are suitable for pharmacological and genetic screening to detect effects on expression of LDLR. The data demonstrate the feasibility of using gene fusions to screen for drugs and genetic factors that affect expression of a wide variety of genes of interest.
