In vivo antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a xenograft model

Abstract

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...

AKT activity regulates the ability of mTOR inhibitors to prevent angiogenesis and VEGF expression in multiple myeloma cells

“…The expression of VEGF in tumor lysate of OPM-2 tumors was also markedly more sensitive to CCI-779, with an ID 50 of B5 mg/kg, while 50% inhibition of VEGF expression in MM tumors with wild-type PTEN was not reached even at the highest dose tested (20 mg/ kg). These data are also consistent with our previous report that PTEN-mutated OPM-2 tumors are significantly more sensitive than PTEN wild-type tumors to the antiangiogenic effects of CCI-779 in vivo (Frost et al, 2004).…”

“…The CCI-779 mTOR inhibitor prevents tumor angiogenesis in an AKT-dependent manner CCI-779 treatment of immunodeficient mice challenged with OPM-2 or 8226 MM cell lines results in tumor regression associated with inhibition of proliferation and angiogenesis, and the induction of apoptosis (Frost et al, 2004). Our initial studies using the U266 cell line transfected with an activated AKT allele indicated that heightened AKT activity sensitizes to the CCI-779 antitumor response in vivo.…”

“…One of the AKT-dependent antitumor effects that we identified was associated with an inhibition of angiogenesis (Frost et al, 2004). Recent work has underscored the importance of angiogenesis in progression of myeloma.…”

“…Confirmation of a true regulatory effect of AKT on sensitivity was obtained when an activated AKT allele was stably transfected into U266 cells. This stably transfected myeloma line was considerably more sensitive in vivo to the antitumor effects of mTOR inhibition than its empty vector transfected control (Frost et al, 2004;Yan et al, 2006).…”

“…In a xenograft model, we showed that the mTOR inhibitor, CCI-779, was an effective anti-myeloma drug, inhibiting in vivo tumor growth of OPM-2, 8226 and U266 MM tumors (Frost et al, 2004;Yan et al, 2006). Interestingly, the level of AKT activity was correlated with antitumor response to CCI-779, with OPM-2, which expresses constitutively active AKT owing to a phosphatase and tensin homolog deleted on chromosome ten (PTEN) mutation (Hyun et al, 2000), being the most sensitive.…”