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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We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways. (Blood.

Purpose Somatic deletions that affect the lymphoid transcription factor–coding gene IKZF1 have previously been reported as independently associated with a poor prognosis in pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). We have now refined the prognostic strength of IKZF1 deletions by analyzing the effect of co-occurring deletions. Patients and Methods The analysis involved 991 patients with BCP ALL treated in the Associazione Italiana Ematologia ed Oncologia Pediatrica–Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 trial with complete information for copy number alterations of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, Xp22.33/Yp11.31 (PAR1 region; CRLF2, CSF2RA, and IL3RA), and ERG; replication of findings involved 417 patients from the same trial. Results IKZF1 deletions that co-occurred with deletions in CDKN2A, CDKN2B, PAX5, or PAR1 in the absence of ERG deletion conferred the worst outcome and, consequently, were grouped as IKZF1plus. The IKZF1plus group comprised 6% of patients with BCP ALL, with a 5-year event-free survival of 53 ± 6% compared with 79 ± 5% in patients with IKZF1 deletion who did not fulfill the IKZF1plus definition and 87 ± 1% in patients who lacked an IKZF1 deletion ( P ≤ .001). Respective 5-year cumulative relapse incidence rates were 44 ± 6%, 11 ± 4%, and 10 ± 1% ( P ≤ .001). Results were confirmed in the replication cohort, and multivariable analyses demonstrated independence of IKZF1plus. The IKZF1plus prognostic effect differed dramatically in analyses stratified by minimal residual disease (MRD) levels after induction treatment: 5-year event-free survival for MRD standard-risk IKZF1plus patients was 94 ± 5% versus 40 ± 10% in MRD intermediate- and 30 ± 14% in high-risk IKZF1plus patients ( P ≤ .001). Corresponding 5-year cumulative incidence of relapse rates were 6 ± 6%, 60 ± 10%, and 60 ± 17% ( P ≤ .001). Conclusion IKZF1plus describes a new MRD-dependent very-poor prognostic profile in BCP ALL. Because current AIEOP-BFM treatment is largely ineffective for MRD-positive IKZF1plus patients, new experimental treatment approaches will be evaluated in our upcoming trial AIEOP-BFM ALL 2017.

In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL.

Dystrophin is selectively localized in the postsynaptic density of neurons in cerebral cortex, hippocampus and cerebellum. Here, we show by double-immunofluorescence staining that dystrophin is extensively colocalized with GABAA receptor subunit clusters in these brain regions. To determine the relevance of this observation, we investigated in mdx mice, which provide a model of Duchenne muscular dystrophy, whether the absence of dystrophin affects the synaptic clustering of GABAA receptors. A marked reduction in the number of clusters immunoreactive for the alpha1 and alpha2 subunits was observed in, respectively, cerebellum and hippocampus of mdx mice, but not in striatum, which is normally devoid of dystrophin. Furthermore, these alterations were not accompanied by a change in gephyrin staining, although gephyrin is colocalized with the majority of GABAA receptor clusters in these regions. These results indicate that dystrophin may play an important role in the clustering or stabilization of GABAA receptors in a subset of central inhibitory synapses. These deficits may underlie the cognitive impairment seen in Duchenne patients.

Key Points Ex vivo drug profiling captures disease-relevant features and relevant sensitivity to therapeutic agents in ALL. A subset of drug-resistant T-ALL without mutations in ABL1 is highly responsive to dasatinib, which provides a rationale for drug repurposing.

TCF3-HLF-fusion positive acute lymphoblastic leukemia (ALL) is currently incurable. Employing an integrated approach, we uncovered distinct mutation, gene expression, and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. Recurrent intragenic deletions of PAX5 or VPREB1 were identified in constellation with TCF3-HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin towards a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics, but sensitivity towards glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.