The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC 50 , 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G 1 cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTENnull tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy. [Cancer Res 2009;69(15):6232-40]

The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors. The mammalian target of rapamycin (mTOR)3 is a central metabolic sensor that coordinates cell growth and proliferation with the availability of growth factors, nutrients, and energy sufficiency. mTOR exists in two multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (1-3). Under conditions of rapid growth and proliferation, mTOR stimulates several anabolic processes, including mRNA translation, transcription, and lipid biosynthesis. The mTORC1 is well known to enhance cap-dependent translation initiation through the direct phosphorylation of S6K1 and 4E-BP1 in response to mitogen and nutrient stimulation (4). The more recently discovered mTORC2 can directly phosphorylate AKT and conventional protein kinase C and is involved in maintenance of actin cytoskeleton in a yet to be defined mechanism. mTOR is a major signaling component of the phosphatidylinositol 3-kinase/AKT pathway that is most frequently dysregulated in cancer (3, 5-7).In addition to the direct control of translational apparatus by mTORC1, several reports have implicated the mTOR signaling in the molecular events occurring the nucleus, such as RNA polymerase (Pol) I transcription, essential for the ribosomal biogenesis and accumulation of cell mass (8 -10). The genetic and biochemical studies in bu...

The mammalian target of rapamycin (mTOR) is a major component of the phosphoinositide 3-kinase (PI3K)/ AKT signaling pathway that is dysregulated in 50% of all human malignancies. Rapamycin and its analogues (rapalogs) partially inhibit mTOR through allosteric binding to mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report WYE-125132 (WYE-132), a highly potent, ATP-competitive, and specific mTOR kinase inhibitor (IC 50 : 0.19 ± 0.07 nmol/L; >5,000-fold selective versus PI3Ks). WYE-132 inhibited mTORC1 and mTORC2 in diverse cancer models in vitro and in vivo. Importantly, consistent with genetic ablation of mTORC2, WYE-132 targeted P-AKT(S473) and AKT function without significantly reducing the steady-state level of the PI3K/PDK1 activity biomarker P-AKT(T308), highlighting a prominent and direct regulation of AKT by mTORC2 in cancer cells. Compared with the rapalog temsirolimus/CCI-779, WYE-132 elicited a substantially stronger inhibition of cancer cell growth and survival, protein synthesis, cell size, bioenergetic metabolism, and adaptation to hypoxia. Oral administration of WYE-132 to tumor-bearing mice showed potent single-agent antitumor activity against MDA361 breast, U87MG glioma, A549 and H1975 lung, as well as A498 and 786-O renal tumors. An optimal dose of WYE-132 achieved a substantial regression of MDA361 and A549 large tumors and caused complete regression of A498 large tumors when coadministered with bevacizumab. Our results further validate mTOR as a critical driver for tumor growth, establish WYE-132 as a potent and profound anticancer agent, and provide a strong rationale for clinical development of specific mTOR kinase inhibitors as new cancer therapy. Cancer Res; 70(2); 621-31. ©2010 AACR.

While small molecule inhibitors of the phosphatidylinositide-3-kinase (PI3K) are expected to impact the development of new cancer therapy, the tumor types and underlying cellular pathways determining inhibitor response remain poorly defined. In this report, we have studied anti-proliferative effects of the PI3K inhibitors WAY-266176 and WAY-266175 in a panel of histologically diverse cancer cells. Inactivation of PI3K caused potent growth suppression in some cells (MDA468, BT549, MDA361, MCF7, LNCap, PC3MM2) but minimal suppression in others (MDA231, MDA435, DU145, HCT116, A549), which correlated with a differential down-regulation of cyclin D1, c-Myc, and induction of apoptosis. A heightened PI3K/AKT/mTOR signaling was linked to the sensitive phenotype but did not generally predict inhibitor response. Interestingly, the resistant cells all displayed an elevated phospho-ERK that remained elevated after serum deprivation. In HCT116 cells, activation mutations in the PI3K catalytic subunit PIK3CA and Ki-Ras correlated with a resistant phenotype, which was partially sensitized by homologous replacement with the wild-type Ki-Ras but not by deletion of cellular PTEN. Depletion of Mek1 via siRNA in resistant cells enhanced PI3K inhibitor-induced growth suppression. Moreover, a profoundly augmented growth suppression and apoptosis were achieved in resistant cells by combination treatment with WAY-266176/WAY-266175 and Mek1 kinase inhibitor CI-1040 or UO126. The combination therapy efficiently inhibited mitogenic signaling and reduced expression of cyclin D1 and c-Myc. Our results identify deregulation of the Ras/Raf/Mek/ERK pathway as a dominant determinant in cancer cell resistance to PI3K inhibitors and highlight combined targeting of PI3K and Mek1 as an effective anticancer strategy.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Metcycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or smallmolecule inhibition leads to de-repression of RXRa resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the epsilon-amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.

The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer.

Within the family of protein kinase C (PKC) molecules, the novel isoform PRKCE (PKCɛ) acts as a bona fide oncogene in in vitro and in vivo models of tumorigenesis. Previous studies have reported expression of PKCɛ in breast, prostate and lung tumors above that of normal adjacent tissue. Data from the cancer genome atlas suggest increased copy number of PRKCE in triple negative breast cancer (TNBC). We find that overexpression of PKCɛ in a non-tumorigenic breast epithelial cell line is sufficient to overcome contact inhibition and results in the formation of cellular foci. Correspondingly, inhibition of PKCɛ in a TNBC cell model results in growth defects in two-dimensional (2D) and three-dimensional (3D) culture conditions and orthotopic xenografts. Using stable isotope labeling of amino acids in a cell culture phosphoproteomic approach, we find that CTNND1/p120ctn phosphorylation at serine 268 (P-S268) occurs in a strictly PKCɛ-dependent manner, and that loss of PKCɛ signaling in TNBC cells leads to reversal of mesenchymal morphology and signaling. In a model of Ras activation, inhibition of PKCɛ is sufficient to block mesenchymal cell morphology. Finally, treatment with a PKCɛ ATP mimetic inhibitor, PF-5263555, recapitulates genetic loss of function experiments impairing p120ctn phosphorylation as well as compromising TNBC cell growth in vitro and in vivo. We demonstrate PKCɛ as a tractable therapeutic target for TNBC, where p120ctn phosphorylation may serve as a readout for monitoring patient response.