A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.Autophagy refers to an evolutionarily conserved process in which intracellular proteins and organelles are sequestered in autophagosomes and subsequently degraded by lysosomal enzymes for the purpose of recycling cellular components to sustain metabolism during nutrient deprivation and to prevent accumulation of damaged proteins and organelles (1, 2). Autophagy is a dynamic process, consisting of several sequential stages (initiation, nucleation, elongation, and maturation) controlled by a group of autophagy-related genes (Atg genes). So far, more than 30 Atg genes have been identified in yeast, and many of them have homologues in mammalian cells (3). Upstream of ATG proteins, mammalian target of rapamycin (mTOR) 4 has been well studied as the key regulatory molecule (4). mTOR is a serine/threonine protein kinase serving as the convergence point for many of the upstream stimuli and pathways to regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis, translation, and autophagy (5-7). Abundance of nutrients, including growth factors, glucose, and amino acids will activate mTOR and suppress autophagy, whereas nutrient deprivation will suppress mTOR, leading to activation of autophagy. At present, the molecular mechanisms downstream of mTOR responsible for its anti-autophagic function have not been fully understood. In yeast, TOR directly targets the ATG13-ATG1 complex and suppresses its function at the initiation stage of autophagy (8). In mammalian cells, the complex containing ULK1 (the ATG1 homologue), ATG13, and FIP200 is directly cont...

More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1a and HIF2a, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2a that allosterically blocks its dimerization with the

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVADinduced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.

Our previous work has shown that autophagy plays a pro-survival function in two necrotic cell death models: zVAD-treated L929 cells as well as H(2)O(2)-treated Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (DKO MEF). This study aims to further explore the regulatory role of autophagy in necrosis by examining the functional role of the phosphoinositide-3 kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway. Our initial intriguing finding was that insulin is able to promote necrotic cell death induced by zVAD and MNNG in L929 cells or by H(2)O(2) in DKO MEF cells cultured in full-growth medium. The pro-necrosis function of insulin was further supported by the observations that insulin is capable of abolishing the protective effect of starvation on necrotic cell death induced by zVAD in L929 cells. Next, we demonstrated that insulin acts on the PI3K-Akt-mTOR pathway to promote necrosis as the suppression of the above pathway by either chemical inhibitors (LY294002 and rapamycin) or mTOR knockdown is able to mitigate the pro-death function of insulin. Finally, we provided evidence that the pro-death function of insulin is dependent on its inhibitory effect on autophagy, which serves as an important pro-survival function in necrosis. Taken together, here we provide compelling evidence to show that activation of the PI3K-Akt-mTOR signaling pathway can promote necrotic cell death via suppression of autophagy, at least in the necrosis models defined in our study in which autophagy serves as a pro-survival function. Data from this study not only further underscore the pro-survival function of autophagy in necrotic cell death, but also provide a novel insight into the intricate connections linking the PI3K-Akt-mTOR signaling pathway with cell death via modulation of autophagy.

The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clinical activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metabolism of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by reduction of phase 2 metabolism. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clinical pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clinical activity shows promise for PT2977 in the treatment of ccRCC.