Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.