Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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

Oncogenic FLT3-ITD supports autophagy via ATF4 in acute myeloid leukemia

International audienceIn acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) account for up to 25% of cases and are associated with a poor outcome. In order to better target this AML subtype, a comprehensive view of how FLT3-ITD impacts AML cell biology is required. Here, we found that FLT3-ITD expression increased basal autophagy in AML cells, and that both pharmacological and genetic inhibition of the receptor reduced autophagy in primary AML samples and cell lines. Conditional expression of shRNAs against key autophagy proteins demonstrated that autophagy is required for AML cell proliferation in vitro and for leukemic cells survival in a mouse model of xenograft. Importantly, autophagy inhibition also overcame FLT3 inhibitor resistance both in vitro and in vivo. The transcription factor ATF4 was identified as an essential actor of FLT3-ITD-induced autophagy. Cellular levels of ATF4 were highly dependent on FLT3-ITD activity, and downregulation of ATF4 inhibited autophagy-dependent AML cell proliferation and improved overall mouse survival similarly to autophagy inhibition. These results suggest that targeting autophagy or ATF4 in patients expressing FLT3 mutations may represent a novel promising and innovative therapeutic strategy for AML