The mammalian target of rapamycin (mTOR) is an important player in the regulation of cell proliferation, growth, differentiation and survival. mTOR regulates both the activity of the 40S ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). This pathway also regulates the translation of mRNAs (e.g. cyclin D1) that are important for the G1/S transition. Inhibition of mTOR pathway increases the rate of turnover of cyclin D1. The cyclin D/CDKs phosphorylate pRb and release the E2Fs which mediate the transcription of proteins required for DNA synthesis, such as dihydrofolate reductase (DHFR). In a variety of cancers, including leukemia and lymphomas the Cyclin D-Cdk4,6/INK4/Rb/E2F pathway is deregulated and contributes to tumor development and also drug resistance towards antifolates. For example, inactivation of p16 and p15 genes, one of the most common genetic events in acute leukemia, is associated with increased DHFR expression and plays an important role in the pathogenesis of acute leukemia. Moreover, the PI3K/Akt/PTEN, the upstream regulators of mTOR pathway are also deregulated in leukemia and lymphomas. Mutations and hemizygous deletions of PTEN have been shown in some primary acute leukemia and non-Hodgkin’s lymphomas, and many hematopoietic cell lines. Akt1 is also frequently translocated in leukemia and lymphoma. Expression of DHFR is regulated both at the transcriptional through the Rb pathway and at the translational levels. We therefore hypothesized that by inhibiting mTOR pathway DHFR levels would be downregulated due to accelerating cyclin D1 degradation or by reducing the levels of DHFR translation through attenuation of 4E-BP1 phosphorylation. To test our hypothesis, four leukemic cell lines were used; namely CEM-S, K562, SKI-DLCL and HL60. The sensitivity of these cell lines against MTX and rapamycin was measured using the XTT assay. Briefly, in each well of a 96-well plate 4000 cells were plated in 300μL of medium containing the desired concentration of drug, and after 5 days, the XTT assay was performed according to standard protocols. After determining the IC50 values of each drug, the sensitivity of these leukemic cells against MTX in the presence of rapamycin at a concentration that corresponds to IC20 of these cell lines with rapamycin alone were determined. The addition of rapamycin sensitized the cells with lymphoblastic origin to MTX approximately 100-fold. We are currently measuring the levels of the proteins involved in the Rb pathway and the mTOR pathway to determine the mechanism of the observed sensitization. Our studies are also underway with combination studies using RAD001, which is a rapamycin analog that is being developed as an antiproliferative agent and other antifolates targeting DHFR and thymidylate synthase.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]