Targeting Deptor in Multiple Myeloma

Abstract 575

DEPTOR is an mTOR binding protein specifically over-expressed in some genetic subtypes of multiple myeloma (MM). DEPTOR binds mTOR in TORC1 and TORC2 complexes and inhibits their kinase activity. As a result, p70 and 4E-BP1 phosphorylation, substrates of TORC1, are decreased. However, TORC1/p70 down-regulation results in marked feedback activation of the PI3K/AKT pathway and AKT becomes activated even though DEPTOR restrains TORC2. Most importantly, DEPTOR knockdown is deleterious to over-expressing MM cell lines, resulting in apoptosis. We hypothesized that compounds preventing DEPTOR-mTOR association would have cytotoxic effects in these cell lines, comparable to DEPTOR knockdown. We, thus, developed a yeast two-hybrid assay where survival of yeast in histidine-depleted media depended upon an association between DEPTOR and mTOR in genetically engineered yeast. After demonstrating a robust yeast two-hybrid protein interaction, yeast were screened against the NCI small inhibitor library (>150,000 compounds) and six compounds were identified that inhibited the interaction. These compounds rapidly increased p70 phosphorylation in MM cell lines in a concentration-dependent fashion, consistent with an inhibition of DEPTOR's inhibitory effect on mTORC1. Co-immunoprecipitation confirmed the compounds inhibited binding of DEPTOR to mTOR without effects on an mTOR-RICTOR interaction. The compounds also significantly inhibited cell survival of cell lines and they were at least 10 × fold more effective against DEPTOR over-expressing MM lines (8226, OPM-2, MM1.S) versus non-expressing lines (U266, FR4, delta 47). These results support a drug development strategy for myeloma that focuses on preventing DEPTOR binding to mTOR within TORC complexes for subsequent anti-myeloma cytotoxic effects.