Linking molecular therapeutics to molecular diagnostics: inhibition of the FRAP/RAFT/TOR component of the PI3K pathway preferentially blocks PTEN mutant cells in vitro and in vivo.

M therapeutics, targeting the underlying defects leading to cancer initiation and progression, are the ‘‘holy grail’’ of cancer research, translation, and therapy. This quest has taken a major leap forward with the demonstration that STI571 (Gleevec) induces clinical remissions in over 90% and molecular remissions in 10–20% of patients with IFN-refractory chronic phase chronic myelogenous leukemia (CML). The efficacy of STI571 in CML has been shown to be caused by the requirement of the bcryabl fusion protein, unique to CML, for its initiation and progression, requiring molecular diagnostics to identify sensitive patients. A plethora of molecular therapeutics targeting signal transduction pathways are under evaluation. Despite the presence of the target in normal cells, the drugs have, in general, been remarkably nontoxic as compared with conventional chemotherapy or radiation therapy (see Fig. 1). As expected, these agents exhibit little, if any, activity in tumors where the target is not amplified or activated. Thus individualization of therapy driven by effective molecular diagnostic approaches to determine the status of the targets in patients’ cancers is as essential a component of a molecular therapeutics program as is the identification and validation of new targets or the development of novel targeted drugs. CCI-779, a homolog of the macrolide antibiotic, rapamycin, which exhibits better pharmacologic characteristics than rapamycin, has been under evaluation as an anticancer agent (1, 2). Although rapamycin and CCI-779 exhibit activity against multiple tumor cell lines in vitro and in vivo, it has not previously been possible to predict which tumors will respond to the effects of rapamycin based on cell lineage or the presence of specific genetic abnormalities. In this issue of PNAS, two manuscripts (3, 4) demonstrate that CCI-779 decreases the growth of tumors containing mutations in the PTEN (a.k.a. TEP and MMAC1) tumor suppressor gene in vitro and in vivo. Thus molecular diagnostics that can identify tumors with abnormalities in PTEN function may predict response to CCI-779, identifying a population of patients likely to benefit from therapy. The effects of CCI-779 on growth of PTEN2y2 embryonic stem cells or on spontaneous tumors in heterozygous PTEN1y2 mice were maintained throughout the course of treatment. Tumor growth, however, resumed after cessation of therapy, indicating that CCI-779 was cytostatic. In addition to inducing a G1 arrest, rapamycin can induce apoptosis, a process that can be linked to the presence of an aberrant G1 checkpoint caused by loss of p53 or p21 function (2, 5). The effects of rapamycin on apoptosis also can be revealed by concurrent treatment with conventional chemotherapy or radiation (6, 7). It may thus be possible to design therapeutic combinations with CCI-779 that will convert the cytostatic effects of CCI-779 to a cytotoxic effect. An alternative paradigm is long-term therapy, converting cancer from a rapidly lethal condition to a chronic disease. Rapamycin and CCI-779 also may demonstrate therapeutic efficacy that is independent of direct tumor cytostasis or apoptosis. At least one cell line, which is resistant to rapamycin in vitro, is sensitive to rapamycin in vivo (7). Further, rapamycin has been demonstrated to inhibit the proliferation of endothelial cells, production of neovascularizing factors, and the production and activation of proteinases, suggesting that rapamycin may inhibit neoangiogenesis or tumor-stromal interactions in vivo (2, 8, 9). Rapamycin and CCI-779 selectively bind FKBP12, inhibiting the activity of the TORy RAFTyFRAP (TOR, target of rapamycin, ref. 10). Inhibition of TOR interferes with the activation of its downstream targets, p70S6 kinase and 4E-BP, resulting in decreased translation of mRNAs with a 59 terminal oligopolypyrimidine tract and CAP-dependent translation, respectively. TOR is a member of the phosphatidylinositol 3-kinase (PI3K) protein super family of serine threonine kinases. This family includes the catalytic subunits of PI3K, the ataxia telangiectasia mutated gene, and the related ATR protein, the PAF400 and TRRAP components of the histone acetylase complex, and the DNA-dependent protein kinase (11, 12). PI3K has the unique additional ability to phosphorylate membrane phosphatidylinositols on the 3 site of the inositol ring. PTEN originally was identified by the late Peter Steck and Ramon Parsons by its localization at a site of loss of heterozygosity and deletions on chromosome 10q23 in human cancers (13, 14). It was simultaneously identified as a transforming growth factor b-regulated gene by Li and Sun (15). PTEN selectively dephosphorylates the same site in membrane phosphatidylinositols phosphorylated by PI3K, an activity that is clearly linked to the tumor suppressor activity of PTEN (11, 12). PTEN also can dephosphorylate proteins and polyphosphoinositols. The roles of the latter processes in the tumor suppressor role of PTEN are less well characterized. The PI3K pathway is constitutively activated in cells with abnormalities in PTEN. Lack of functional PTEN results in a cell autonomous accumulation of phosphatidylinositol 3,4,5P3, which leads to the recruitment to the membrane and activation of a subset of pleckstrin homology, Phox, and C1 and C2 domain-containing proteins (11, 12). Introduction of a wild-type PTEN gene into cancer cell lines lacking functional PTEN protein decreases signaling through the PI3K pathway as indicated by a decrease in phosphatidylinositol 3,4,5P3 levels and alterations in downstream events including activity, phosphorylation, or localization of