Unlike Inactivating PTEN Mutations, Pharmacological Inhibition of PTEN Does Not Increase the Sensitivity of Multiple Myeloma Cells to Inhibition of the PI3K/Akt/mTOR Pathway.

Abstract

PTEN-deficient multiple myeloma (MM) and other tumor cell lines have been shown to be remarkably sensitive to inhibitors of the PI3K/Akt/mTOR pathway (Shi Y, et al. Cancer Res 2002; Simth Y, et al. Proc Am Assoc Cancer Res 2002; Neshat MS, et al. Proc. Natl. Acad. Sci. USA 2001.). Inactivating mutations of PTEN, however, are very rare in human MM cells. We investigated whether pharmacological inhibition of wild type PTEN might also sensitize MM cells to inhibitors of PI3K and mTOR, and therefore provide the rationale for novel therapeutically useful combinations. We tested the effect of the PTEN antagonist, SF1740 (Semafore Inc, Indianapolis, IN), in combination with the mTOR inhibitor, rapamycin, or the PI3K inhibitor, SF1101, (also known as LY294002, from Semafore Inc, Indianapolis, IN) in U266, MM1R, MM1S myeloma cells. Using the Malachite Green assay, the IC50 for PTEN inhibition by SF1740 was 0.4 μM, and maximal inhibition was achieved at a concentration of 1μM. We treated MM cells with rapamycin (1, 5, 10 nM) or SF1101 (10, 25, 50 μM) alone or in combination with SF1740 for 24, 48, 72 and 96 hours. Cellular toxicity was assessed using the MTT assay, apoptosis by annexin V/PI staining, and the effect on intracellular signaling was monitored by Western blotting. SF1740 (1 μM) alone increased cell growth, and failed to sensitize cells to the inhibitory effects of rapamycin (1–10 nM) at all time points. There were no significant differences in cell survival between the combination of the two drugs and rapamycin alone when treated from 48–96hr. Cytotoxicity was unaffected by the sequence of drug treatment; concurrent or 24-hour pre-treatment with SF1740. Similar results were obtained using the combination of SF1740 (1 μM) and SF1101 used at concentrations of 10, 25, and 50 μM, with no difference in MM cell inhibition and survival between the SF1101 and the combination with SF1740. We conclude that in MM cells, pharmacological inhibition of wild type PTEN does not appear to replicate the effect of inactivating mutations of PTEN in increasing cell sensitivity to inihibitors of the PI3K/Akt/mTOR pathway. The results do not support the clinical use of the PTEN inhibitor SF1740 in combination with either PI3K or mTOR inhibitors. Xiaojing Wang and Shuhong Zhang contributed equally to this work.