Autophagy plays a critical role in cell metabolism by degrading and recycling internal components when challenged with limited nutrients. This fundamental and conserved mechanism is based on a membrane trafficking pathway in which nascent autophagosomes engulf cytoplasmic cargo to form vesicles that transport their content to the lysosome for degradation. Based on this simple scheme, autophagy modulates cellular metabolism and cytoplasmic quality control to influence an unexpectedly wide range of normal mammalian physiology and pathophysiology. In this review, we summarise recent advancements in three broad areas of autophagy regulation. We discuss current models on how autophagosomes are initiated from endogenous membranes. We detail how the uncoordinated 51-like kinase (ULK) complex becomes activated downstream of mechanistic target of rapamycin complex 1 (MTORC1). Finally, we summarise the upstream signalling mechanisms that can sense amino acid availability leading to activation of MTORC1.

Autophagy maintains metabolism in response to starvation, but each nutrient is sensed distinctly. Amino acid deficiency suppresses mechanistic target of rapamycin complex 1 (MTORC1), while glucose deficiency promotes AMP-activated protein kinase (AMPK). The MTORC1 and AMPK signaling pathways converge onto the ULK1/2 autophagy initiation complex. Here, we show that amino acid starvation promoted formation of ULK1- and sequestosome 1/p62-positive early autophagosomes. Autophagosome initiation was controlled by MTORC1 sensing glutamine, leucine, and arginine levels together. In contrast, glucose starvation promoted AMPK activity, phosphorylation of ULK1 Ser555, and LC3-II accumulation, but with dynamics consistent with a block in autophagy flux. We studied the flux pathway and found that starvation of amino acid but not of glucose activated lysosomal acidification, which occurred independently of autophagy and ULK1. In addition to lack of activation, glucose starvation inhibited the ability of amino acid starvation to activate both autophagosome formation and the lysosome. Activation of AMPK and phosphorylation of ULK1 were determined to specifically inhibit autophagosome formation. AMPK activation also was sufficient to prevent lysosome acidification. These results indicate concerted but distinct AMPK-dependent mechanisms to suppress early and late phases of autophagy.

There has been long-standing interest in targeting pro-survival autophagy as a combinational cancer therapeutic strategy. Clinical trials are in progress testing chloroquine (CQ) or its derivatives in combination with chemo- or radiotherapy for solid and haematological cancers. Although CQ has shown efficacy in preclinical models, its mechanism of action remains equivocal. Here, we tested how effectively CQ sensitises metastatic breast cancer cells to further stress conditions such as ionising irradiation, doxorubicin, PI3K-Akt inhibition and serum withdrawal. Contrary to the conventional model, the cytotoxic effects of CQ were found to be autophagy-independent, as genetic targeting of ATG7 or the ULK1/2 complex could not sensitise cells, like CQ, to serum depletion. Interestingly, although CQ combined with serum starvation was robustly cytotoxic, further glucose starvation under these conditions led to a full rescue of cell viability. Inhibition of hexokinase using 2-deoxyglucose (2DG) similarly led to CQ resistance. As this form of cell death did not resemble classical caspase-dependent apoptosis, we hypothesised that CQ-mediated cytotoxicity was primarily via a lysosome-dependent mechanism. Indeed, CQ treatment led to marked lysosomal swelling and recruitment of Galectin3 to sites of membrane damage. Strikingly, glucose starvation or 2DG prevented CQ from inducing lysosomal damage and subsequent cell death. Importantly, we found that the related compound, amodiaquine, was more potent than CQ for cell killing and not susceptible to interference from glucose starvation. Taken together, our data indicate that CQ effectively targets the lysosome to sensitise towards cell death but is prone to a glucose-dependent resistance mechanism, thus providing rationale for the related compound amodiaquine (currently used in humans) as a better therapeutic option for cancer.

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1-ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1-ULK1 signalling module.

Tumor Associated Macrophages (TAMs), characteristic of an M2-like immune-suppressive phenotype, can induce proliferation and survival of tumor cells, facilitate angiogenesis, and suppress anti-tumor immune responses via expression of co-inhibitory molecules (e.g. PD-L1) and cytokines (e.g. IL-10, TGF-β). TAMs are therefore a highly attractive target of innovative cancer immunotherapies. Understanding the ability of pre-clinical candidate compounds to reverse TAM (M2-like)-mediated immune suppression and the potential for reprogramming of M2-like macrophages to a M1-like phenotype is key in the development of effective TAM-targeted cancer immunotherapies. Here we outline development of an assay to assess the capabilities of pre-clinical compounds to reverse M2 macrophage-mediated immune suppression. Monocytes were isolated from whole blood obtained from healthy volunteers and cultured under M2-polarising conditions. The resulting macrophages were phenotypically characterized and then used in co-culture with autologous PBMC, stimulated through T cell receptor ligation. Resulting cytokine production was assessed, alongside CD4+ and CD8+ T cell viability and cell cycle status (flow cytometry). M2-like macrophages polarized with M-CSF displayed an immune suppressive phenotype as shown by their production of IL-10 and their inhibition of IFN-γ production by, and cell cycle status of, T cells in co-culture assays. This suppressive activity was only partially reversed by PD-1-blockade. Modifications to the macrophage polarization protocol were seen to alter the resulting macrophage cell surface phenotype (e.g. expression levels of PD-L1, TIM-3 and CD200R) and their suppressive activity in the co-culture assay. These cell surface phenotypes broadly reflected those seen amongst tumor-derived macrophages from renal and ovarian carcinoma patients (CD14+ CD163+ with expression of TIM-3 and LAG-3). Moreover, compound-mediated changes in functionality seen with macrophages polarized from healthy PBMC monocytes could also be seen using patient-derived material. The assay outlined here therefore provides a M-o-A human in vitro system to test novel compound activity (either singly or in combination) upon TAM-like macrophage generation, phenotype and suppressive function. This allows selection of the most efficacious compounds for further investigation using patient-derived immune cells. Citation Format: Laura E. Gallagher, Andrew Hall, Lauren A. Patience, Lucia Janicova, Stephen Anderton. Interrogation and modulation of the immunosuppressive activity of human TAM-like macrophages using in vitro cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3808.