Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin and CCI-779, have shown preclinical potential as therapy for multiple myeloma. By inhibiting expression of cell cycle proteins, these agents induce G 1 arrest. However, by also inhibiting an mTOR-dependent serine phosphorylation of insulin receptor substrate-1

In vitro studies indicate the therapeutic potential of mTOR inhibitors in treating multiple myeloma. To provide further support for this potential, we used the rapamycin analog CCI-779 in a myeloma xenograft model. CCI-779, given as 10 intraperitoneal injections, induced significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 cell lines. Effective doses of CCI-779 were associated with modest toxicity, inducing only transient thrombocytopenia and leukopenia. Immunohistochemical studies demonstrated the antitumor responses were associated with inhibited proliferation and angiogenesis, induction of apoptosis, and reduction in tumor cell size. Although CCI-779-mediated inhibition of the p70 mTOR substrate was equal in 8226 and OPM-2 tumor nodules, OPM-2 tumor growth was considerably more sensitive to inhibition of proliferation, angiogenesis, and induction of apoptosis. Furthermore, the OPM-2 tumors from treated mice were more likely to show downregulated expression of cyclin D1 and c-myc and up-regulated p27 expression.Because earlier work suggested heightened AKT activity in OPM-2 tumors might induce hypersensitivity to mTOR inhibition, we directly tested this by stably transfecting a constitutively active AKT allele into U266 cells. The in vivo growth of the latter cells was remarkably more sensitive to CCI-779 than the growth of control U266 cells. IntroductionThe phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) signaling pathway is important for the survival and growth of multiple myeloma (MM) cells and is an attractive target for antitumor therapy. [1][2][3] An important downstream target of PI3-K/AKT is the mammalian target of rapamycin (mTOR), which mediates phosphorylation of p70S6 kinase (p70) and 4E-BP1, 4 proteins responsible for the translation and expression of D-type cyclins and c-myc. 5,6 By preventing these phosphorylation events, mTOR inhibitors down-regulate such expression and induce G 1 cell cycle arrest. 7 In addition, these drugs up-regulate expression of the p27 CDK inhibitor, which may also contribute to G 1 arrest. 8 The in vitro sensitivity of MM cells to the antitumor effects to mTOR inhibitors frequently correlates with heightened AKT activity. [9][10][11] Rapamycin is a classical mTOR inhibitor. The poor solubility that compromised rapamycin as an intravenous agent led to the development of a more soluble ester analog of rapamycin, CCI-779. 12 We have shown in vitro anti-MM activity of rapamycin and CCI-779. 9,11,13 Exposure to these mTOR inhibitors prevents the proliferation of PTEN-and RAS-mutated myeloma cell lines and of interleukin-6 (IL-6)-stimulated proliferation of nonmutated myeloma clones. To provide a further preclinical rationale for the development of mTOR inhibitors in patients, we initiated the current study testing the effects of CCI-779 in vivo against human MM tumor growth in a murine xenograft model. Our results confirm that CCI-779 is effective in vivo against myeloma cells and demonstrate inhibited proliferation, angiogenes...

Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress-inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib.