In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Novel therapeutic strategies are needed to address the emerging problem of imatinib resistance. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) is being evaluated for imatinib-resistant chronic myelogenous leukemia (CML) and has multiple cellular effects, including the induction of autophagy and apoptosis. Considering that autophagy may promote cancer cell survival, we hypothesized that disrupting autophagy would augment the anticancer activity of SAHA. Here we report that drugs that disrupt the autophagy pathway dramatically augment the antineoplastic effects of SAHA in CML cell lines and primary CML cells expressing wild-type and imatinib-resistant mutant forms of BcrAbl, including T315I. This regimen has selectivity for malignant cells and its efficacy was not diminished by impairing p53 function, another contributing factor in imatinib resistance. IntroductionImatinib (Gleevec; STI-571), a targeted competitive inhibitor of the Bcr-Abl tyrosine kinase, revolutionized the clinical treatment of chronic myelogenous leukemia (CML). 1 However, acquired imatinib resistance during the accelerated and blast crisis phases of the disease is an emerging problem and has been linked to gene amplification, to point mutations in Bcr-Abl that impede drug binding or structurally preclude adoption of the inactive conformation, and to loss of p53 function. [2][3][4] Two novel inhibitors of Bcr-Abl, dasatinib and nilotinib, have been evaluated to address this problem. 5,6 Both agents produce clinical responses in many imatinib-refractory patients but not in those carrying the most drug-resistant T315I mutation, which confers cross-resistance to nilotinib and dasatinib. 7,8 The lack of effective therapeutic regimens for T315I patients thus highlights the dire need for novel therapeutic strategies that are effective in treating these patients.Histone deacetylase (HDAC) inhibitors represent a novel class of anticancer agents currently under investigation in preclinical models and in phase 1/2 clinical trials. [9][10][11][12] Suberoylanilide hydroxamic acid (SAHA) is an orally bioavailable, well-tolerated pan-HDAC inhibitor with anticancer activity in hematologic and solid malignancies. 12,13 SAHA's anticancer effects have been linked to the generation of reactive oxygen species (ROS) and to the induction of apoptosis, growth arrest, polyploidy, and autophagy. 14-17 Whether SAHA's ability to augment autophagy affects its anticancer activity remains unclear. Here we tested the hypotheses that disruption of the autophagy pathway would significantly enhance the anticancer activity of SAHA and that this would prove effective in killing imatinib-resistant CML. Patients, materials, and methods Cells and cell cultureBa/F3 cells and Ba/F3 cells engineered to express comparable levels of wild-type (p210) and mutant forms of Bcr-Abl (E255K, M351T, and T315I) were maintained as previously described. 2 K562 and LAMA 84 CML cells were maintained in RPMI-1640 media with 10% heat-inactivated fetal bovine serum at 3...

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (ρ-) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.

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Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH 2 -terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca 2+ levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin. (Cancer Res 2005; 65(24): 11658-66)

NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models.