Suppl fig 2:A) Co-IP between DEPTOR & mTOR in 8226 treated with NSC126405 or 3g; B,C&D) Immunoblot of 8226 cells after 6 hrs incubation with drugs. Relative ex- pression below gels are means of 3 separate experiments; E) Bead pull-down assay to quantify binding of recombinant DEPTOR to mTOR (see Materials & Methods); Lane 1 (furthest to the left) is a control showing anti-FLAG-containing beads (w/o conjugation to mTOR-FLAG) do not non-specifically bind to GST-DEPTOR while mTOR-FLAG-conjugated beads successfully bind GST-DEPTOR (lane 2); Below gel are results from 3 separate experiments showing mean +/-SD percent of control, n=3. DM=DMSO treatment; Drug J007 is a myc IRES inhibitor (6) of similar size to 3g and used as an additional negative control. *=significant decrease from control (DMSO), p<0.05; **=p<0.01. F) Immunoblot after incubation with drug 3g (upper) or NSC126405 (lower).
ARTICLE ABSTRACT
Prior work indicates DEPTOR expression in multiple myeloma cells could be a therapeutic target. DEPTOR binds to mTOR via its PDZ domain and inhibits mTOR kinase activity. We previously identified a drug, which prevented mTOR–DEPTOR binding (NSC126405) and induced multiple myeloma cytotoxicity. We now report on a related therapeutic, drug 3g, which induces proteasomal degradation of DEPTOR. DEPTOR degradation followed drug 3g binding to its PDZ domain and was not due to caspase activation or enhanced mTOR phosphorylation of DEPTOR. Drug 3g enhanced mTOR activity, and engaged the IRS-1/PI3K/AKT feedback loop with reduced phosphorylation of AKT on T308. Activation of TORC1, in part, mediated multiple myeloma cytotoxicity. Drug 3g was more effective than NSC126405 in preventing binding of recombinant DEPTOR to mTOR, preventing binding of DEPTOR to mTOR inside multiple myeloma cells, in activating mTOR and inducing apoptosis in multiple myeloma cells. In vivo, drug 3g injected daily abrogated DEPTOR expression in xenograft tumors and induced an antitumor effect although modest weight loss was seen. Every-other-day treatment, however, was equally effective without weight loss. Drug 3g also reduced DEPTOR expression in normal tissues. Although no potential toxicity was identified in hematopoietic or hepatic function, moderate cardiac enlargement and glomerular mesangial hypertrophy was seen. DEPTOR protected multiple myeloma cells against bortezomib suggesting anti-DEPTOR drugs could synergize with proteasome inhibitors (PI). Indeed, combinations of drug NSC126405 + bortezomib were synergistic. In contrast, drug 3g was not and was even antagonistic. This antagonism was probably due to prevention of proteasomal DEPTOR degradation.
