The LCLs have limitations, as we have previously discussed (Niu et al., 2010). For example, EBV transformation can induce chromosomal instability and cellular changes (Sie et al., 2009). In addition, other factors such as cell growth rate and ATP level can have an effect on cytotoxicity (Choy et al., 2008). Since these cell lines do not necessarily represent the response of other types of tissues or cells (Dimas et al., 2009), we selected the top candidate genes based on our analyses to determine their contribution to variation in response to mTOR inhibitors. Two clinically relevant tumor cell lines, renal carcinoma (Caki2) and glioblastoma (U87), were selected for functional validation (Supplementary Figures S2, S3) since mTOR inhibitors are used as a treatment for these two types of tumors (Pantuck et al., 2006; Brugarolas et al., 2008; Cloughesy et al., 2008) and because our data suggested that these two cell lines were relatively more sensitive to mTOR inhibitor treatment. A fibroblast cell line (IMR90) was also included as a comparison to the tumor cell lines (Supplementary Figure S4). The two tumor cell lines, Caki2 and U87, tended to show similar results for several of the genes tested: ECOP, MGLL, and MAN1B. Our study showed that knockdown of these genes sensitized both Caki2 and U87 cells to mTOR inhibitors. ECOP (EGFR-coamplified and overexpressed protein), a gene which is amplified and overexpressed in at least a third of glioblastomas with EGFR amplification (Eley et al., 2002), is known to be a key regulator of NF-κB transcriptional activity that can contribute to cell survival (Park and James, 2005). IMR90 cells, on the other hand, seemed to be impacted by a different panel of genes, BTG2, FBXW7, NDUFAF2, PHLDA1, and DMD, whose knockdown did not have a significant impact in the two tumor cell lines, suggesting cell line-specific effects. Many of these genes have not been previously reported to interfere with the mTOR pathway except for FBXW7 (F-box and WD repeat domain containing 7), which is known to target mTOR for degradation and which cooperates with PTEN for tumor suppression (Mao et al., 2008), and BTG2 (B-cell translocation gene 2), which has been reported to inhibit AKT phosphorylation and mTOR signaling. Our results were compatible with the conclusion that down-regulation of FBXW7 restored the target for mTOR inhibitors, thus sensitizing cells to mTOR inhibitors, while knockdown of BTG2 activated the mTOR pathway which might cause the cells to become “addicted” to the mTOR pathway and, therefore, to benefit from mTOR inhibition. It is also worth noting that knockdown of ZNF765 (zinc finger protein 765) was found to sensitize cells to mTOR inhibitors in both the IMR90 and U87 cell lines (Table 2). ZNF765 is located on chromosome 19 and little is known with regard to its function. Therefore, its involvement in the mTOR pathway and response to mTOR inhibitors needs to be investigated further in the future.