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]

Autophagy is a tightly regulated catabolic process that plays key roles in normal cellular homeostasis and survival during periods of extracellular nutrient limitation and stress. The environmental signals that regulate autophagic activity are only partially understood. Here, we report a direct link between glutamine (Gln) metabolism and autophagic activity in both transformed and nontransformed human cells. Cells cultured for more than 2 days in Gln-containing medium showed increases in autophagy that were not attributable to nutrient depletion or to inhibition of mammalian target of rapamycin. Conditioned medium from these cells contained a volatile factor that triggered autophagy in secondary cell cultures. We identified this factor as ammonia derived from the deamination of Gln by glutaminolysis. Gln-dependent ammonia production supported basal autophagy and protected cells from tumor necrosis factor-alpha (TNF-alpha)-induced cell death. Thus, Gln metabolism not only fuels cell growth but also generates an autocrine- and paracrine-acting regulator of autophagic flux in proliferating cells.

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic lossof-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagyrelated 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.M acroautophagy is a catabolic pathway that shuttles cytoplasmic components via double-membrane vesicles (autophagosomes) into lysosomes for degradation and recycling. Autophagosome formation and elongation are facilitated by ubiquitin-like molecules such as MAP1LC3A/B (herein referred to as "LC3") and its homologs which are directly conjugated to phosphatidylethanolamine (PE), a reaction which requires the ubiquitin E1-like activity of autophagy-related 7 (ATG7), the E2-like activity of ATG3, and the E3-like activity of the ATG5-ATG12-ATG16L1 complex (1). Autophagy cargo receptors such as p62/ SQSTM1 bind both LC3 and ubiquitinated cargo, enabling cargo recruitment into autophagosomes and delivery to lysosomes (2, 3).Basal levels of macroautophagy control cellular homeostasis by clearing misfolded proteins or damaged organelles (4, 5). Upon starvation, macroautophagy can be induced above basal levels to supply the cell with nutrients (6, 7). This prosurvival function of macroautophagy is also used by cancer cells under conditions of metabolic stress (8). However, the role of autophagy in cancer is complex and context dependent, because the pathway has been reported to have tumor-suppressing as well as tumor-promoting properties (9-11). Liver-specific deletion of ATG7 results in increased formation of liver tumors through the activation of the Nrf2 pathway (12). Furthermore, the essential autophagy component beclin-1 inhibits tumorigenesis of breast carcinoma cells, and monoallelic deletion of beclin-1 is associated with an enhanced risk of breast cancer (13-15). I...

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC 80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADCresistant cell models retain sensitivity to other ADCs or standardof-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance.

The stability of the connection between the antibody and the toxin can have a profound impact on ADC safety and efficacy. There has been increasing evidence in recent years that maleimide-based ADCs are prone to payload loss via a retro-Michael type reaction. Herein, we report a mild method for the hydrolysis of the succinimide-thioether ring which results in a "ring-opened" linker. ADCs containing this hydrolyzed succinimide linker show equivalent cytotoxicity, improved in vitro stability, improved PK exposure, and improved efficacy as compared to their nonhydrolyzed counterparts. This method offers a simple way to improve the stability, exposure, and efficacy of maleimide-based ADCs.