Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.

/pdf/ampk-and-mtor-regulate-autophagy-through-direct-14wqsz16ke.pdf

AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1

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 flux 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, 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. 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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. 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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Methods in Mammalian Autophagy Research

The clinical use of anthracyclines like doxorubicin and daunorubicin can be viewed as a sort of double-edged sword. On the one hand, anthracyclines play an undisputed key role in the treatment of many neoplastic diseases; on the other hand, chronic administration of anthracyclines induces cardiomyopathy and congestive heart failure usually refractory to common medications. Second-generation analogs like epirubicin or idarubicin exhibit improvements in their therapeutic index, but the risk of inducing cardiomyopathy is not abated. It is because of their janus behavior (activity in tumors vis-a-vis toxicity in cardiomyocytes) that anthracyclines continue to attract the interest of preclinical and clinical investigations despite their longer-than-40-year record of longevity. Here we review recent progresses that may serve as a framework for reappraising the activity and toxicity of anthracyclines on basic and clinical pharmacology grounds. We review 1) new aspects of anthracycline-induced DNA damage in cancer cells; 2) the role of iron and free radicals as causative factors of apoptosis or other forms of cardiac damage; 3) molecular mechanisms of cardiotoxic synergism between anthracyclines and other anticancer agents; 4) the pharmacologic rationale and clinical recommendations for using cardioprotectants while not interfering with tumor response; 5) the development of tumor-targeted anthracycline formulations; and 6) the designing of third-generation analogs and their assessment in preclinical or clinical settings. An overview of these issues confirms that anthracyclines remain "evergreen" drugs with broad clinical indications but have still an improvable therapeutic index.

/pdf/anthracyclines-molecular-advances-and-pharmacologic-4vygbx1i0b.pdf

Anthracyclines: Molecular Advances and Pharmacologic Developments in Antitumor Activity and Cardiotoxicity

The role of senescence in the action of antitumor drugs.

Background: Mediastinal irradiations (XRT) in cancer treatment are associated with development of cardiomyopathy. Radiation-induced cardiomyopathy is characterized by a latent phase. In mice, an impaired contractile reserve arises early after XRT despite a normal resting cardiac function. XRT induces interleukin-1 (IL-1) in the heart and lungs. We propose to investigate the mechanistic role of IL-1 in the development of XRT-induced cardiomyopathy.

Methods: We irradiated (14Gy, single dose) the thorax of C57BL/6J wild type (wt) mice or IL-1 receptor type I knock out mice (IL-1RI KO, which are not responsive to IL-1)(N=12/group). Wt- and KO-sham irradiated mice were used as controls. The serum levels of cardiac Troponin I (cTnI) were measured by ELISA 3 days after XRT and RT-PCR was used to measure IL-1 beta (IL-1β) mRNA. Left ventricular (LV) ejection fraction (EF) was evaluated by echocardiography at baseline and 3 days following XRT. The beta-adrenergic receptor agonist isoproterenol (10 ng/mouse) was used to measure the contractile reserve (LVEF change).

Results: XRT induced IL-1β mRNA in wt compared to sham-irradiated mice (data not shown). Serum levels of cTnI after XRT were significantly lower in KO vs wt mice (1.04±0.4 ng/ml KO vs 4.6±1.5 ng/ml wt, P=0.037, Figure A). Resting LVEF at 3 days was unchanged in both wt and IL-1RI KO mice after XRT, whereas contractile reserve was significantly reduced in wt but not in KO mice (+16.0±4.1% wt vs +28.6±3.0% KO; P=0.046, Figure B).

Conclusions: IL-1 amplifies myocardial damage during the acute phase of XRT-induced cardiomyopathy. IL-1 blockade may represent a novel therapeutic strategy to protect the heart following XRT. . 

![Graphic][1] [1]: /embed/inline-graphic-1.gif

Abstract 17799: Role Of Interleukin-1 In Acute Radiation-induced Cardiomyopathy

Cell size and growth are tightly regulated processes balancing synthesis and metabolism to ensure proper cell function. Deviations from normal growth in response to drug treatment provide insights into the induced cellular dysfunction caused by pharmacotherapy. These changes in biomass growth have shown promise as a marker for drug sensitivity. However, like many biomarkers, the output cannot be treated as binary. Just as cancer cells are heterogenous on the molecular level, individual cells’ biomass response to drug treatment can range from cell death to no effect. It is therefore important to begin to survey for the full range of biomass growth responses to drug treatment to understand the dysfunction induced. Here, we explore the response of different cancer cell lines to treatments that induce biomass growth changes ranging from apoptosis to senescence to simply delayed regrowth. These longer-term studies, ranging up to six days of constant monitoring, aid in the interpretation of more commonly performed shorter-term biomass growth experiments and identify cells of interest for further molecular characterization in these cell lines.

The spectrum of single cell drug response through the lens of long-term high-speed live cell interferometry

Anti-estrogens or aromatase inhibitors in combination with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are the current standard of care for estrogen receptor-positive (ER+) Her-2 negative metastatic breast cancer. Although these combination therapies prolong progression-free survival compared to endocrine therapy alone, the growth-arrested state of residual tumor cells is clearly transient. Tumor cells that escape what might be considered a dormant or quiescent state and regain proliferative capacity often acquire resistance to further therapies. Our studies are based upon the observation that breast tumor cells arrested by Fulvestrant + Palbociclib enter into states of both autophagy and senescence from which a subpopulation ultimately escapes, potentially contributing to recurrent disease. Autophagy inhibition utilizing pharmacologic or genetic approaches only moderately enhanced the response to Fulvestrant + Palbociclib in ER+ MCF-7 breast tumor cells, slightly delaying proliferative recovery. In contrast, the BET inhibitor/degrader, ARV-825, prolonged the growth arrested state in both p53 wild type MCF-7 cells and p53 mutant T-47D cells and significantly delayed proliferative recovery. In addition, ARV-825 added after the Fulvestrant + Palbociclib combination promoted apoptosis and demonstrated efficacy in resistant RB deficient cell lines. These studies indicate that administration of BET inhibitors/degraders, which are currently being investigated in multiple clinical trials, may potentially improve standard of care therapy in metastatic ER+ breast cancer patients and may further prolong progression-free survival.

/pdf/the-bet-inhibitor-degrader-arv-825-prolongs-the-growth-27c1857i.pdf

The BET inhibitor/degrader ARV-825 prolongs the growth arrest response to Fulvestrant + Palbociclib and suppresses proliferative recovery in ER-positive breast cancer

The Vinca alkaloid vincristine and the epipodophyllotoxin derivative teniposide significantly elevate steady state levels of methotrexate in the Ehrlich ascites tumor cell in vitro . However, the presence of circulating serum proteins compromises this drug-cell interaction. In the presence of bovine serum albumin, elevation of cellular methotrexate levels by vincristine and teniposide in vitro is reduced by 60–70%. In the presence of physiological concentrations of human serum albumin and human serum glycoproteins, the enhancement of cellular methotrexate accumulation by vincristine is reduced by approximately 50% while enhancement of cellular methotrexate accumulation by teniposide is essentially eliminated. These findings may in part explain the observations that vincristine does not alter the cellular pharmacokinetics of methotrexate in vivo and may provide a rationale for the lack of therapeutic synergism between vincristine and methotrexate when the Vinca alkaloid is administered concurrent with or prior to the antifolate compound in the tumor bearing animal. Furthermore these studies indicate the difficulties inherent in attempting to extrapolate from in vitro studies of drug-cell interaction to the intact animal.

https://cancerres.aacrjournals.org/content/canres/45/12_Part_1/6290.full.pdf

Serum Protein Reduction of the Enhancement of Methotrexate Accumulation by Vincristine and 4′-Demethylepipodophyllotoxin in the Ehrlich Ascites Tumor Cell in Vitro

David A. Gewirtz

Papers

The role of senescence in the action of antitumor drugs.

Abstract 17799: Role Of Interleukin-1 In Acute Radiation-induced Cardiomyopathy

The spectrum of single cell drug response through the lens of long-term high-speed live cell interferometry

The BET inhibitor/degrader ARV-825 prolongs the growth arrest response to Fulvestrant + Palbociclib and suppresses proliferative recovery in ER-positive breast cancer

Serum Protein Reduction of the Enhancement of Methotrexate Accumulation by Vincristine and 4′-Demethylepipodophyllotoxin in the Ehrlich Ascites Tumor Cell in Vitro