Macroautophagy (autophagy) is a process whereby cellular components such as proteins and organelles are captured within autophagosomes, and are delivered to the lysosomes for degradation 
. Autophagy is activated by starvation, enabling cellular and mammalian viability by providing an internal source of building blocks for macromolecular synthesis. This sustains biosynthetic and energy requirements during interruptions in nutrient availability 
. Cellular damage also activates autophagy, which promotes cell survival by preventing accumulation of damaged proteins and organelles, particularly mitochondria that are a source of oxidative stress 
. These intracellular recycling and garbage disposal roles of autophagy provide cells with the inherent flexibility to cope with periods of deprivation while limiting oxidative stress. Although autophagy is important for the function and survival of normal cells, it is also utilized by tumor cells and may be therapeutically counterproductive 
Metabolic stress due to oxygen, nutrient and growth factor deprivation is common in tumors due to insufficient vascularization, and autophagy is activated in tumor cells in hypoxic regions to support survival 
. Tumor cells in oxygen-deprived hypoxic regions are more resistant to treatment and this autophagy-conferred survival advantage has led to the realization that autophagy inhibitors may be useful to improve cancer therapy 
. Moreover, cytotoxic and targeted cancer therapeutics induce autophagy, either by infliction of stress and cellular damage or by inhibition of signaling pathways mimicking deprivation 
. The potential of environmental and therapeutic autophagy induction to limit treatment effectiveness remains to be addressed.
Cells respond to growth signals in their environment through the phosphoinositide 3-kinase (PI3K) pathway and regulation of the serine-threonine kinase mTOR. mTOR promotes cell growth in response to nutrient and growth factor availability, while suppressing autophagy 
. In nutrient and growth factor replete conditions, the PI3K pathway activates mTOR through the mTORC1 complex that binds, phosphorylates and inhibits key autophagy machinery components required to initiate autophagosome formation 
. Tumor cells commonly take advantage of the cell growth-promoting function of the PI3K pathway by acquiring mutations that result in its constitutive activation 
. As such, inhibitors of the PI3K pathway are potentially useful for restricting tumor growth, although they also activate autophagy. It is unclear, however, if this induction of autophagy is a mechanism of resistance by increasing tumor cell stress tolerance.
mTOR inhibitors include both allosteric (rapamycin/sirolimus, and other rapalogs such as temsirolimus and everolimus) and ATP-competitive mechanistic classes. Both temsirolimus and everolimus have shown efficacy in the treatment of renal cancer and are approved by the Food and Drug Administration (FDA) to treat RCC 
. In the first line setting, temsirolimus improves overall survival in RCC patients with metastatic disease. Unfortunately, all patients eventually relapse, contributing to the dismal prognosis of this devastating disease. It is expected that most targeted therapeutics, including temsirolimus, will require combination therapy for improved therapeutic outcome, necessitating identification of synthetic lethal pathways.
If autophagy induction by mTOR inhibitors promotes tumor cell survival, then combination treatment with an autophagy inhibitor may be expected to promote tumor cell death 
. One tractable, small molecule approach to autophagy inhibition is the lysosomotropic anti-malaria drug CQ and its analogues. CQ prevents lysosome acidification and thereby the degradation of the products of autophagy, resulting in autophagolysosome accumulation. In a mouse model of c-Myc-driven lymphoma, a CQ analogue hydroxychloroquine (HCQ) promotes tumor cell death by either p53 activation or alkylating agents 
. In mouse models for ataxia telangiectasia and Burkitt's lymphoma, CQ suppresses spontaneous tumorigenesis 
. CQ also has antitumor activity in mouse models of BRC-Abl leukemia 
and in combination with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) in a mouse model and in human samples of CML 
. In human solid tumor xenografts, CQ inhibits tumor growth as a single agent in pancreatic cancer, and in combination with PI3K pathway inhibitors in glioma or with mTOR inhibitors in tuberous sclerosis complex (TSC)-deficient tumors 
. These preclinical studies support the approach of autophagy inhibition in cancer therapy 
. One major limitation is the unknown mechanism by which autophagy inhibition promotes cancer cell death, particularly in combination with PI3K pathway inhibition, and what defines cancer susceptibility to autophagy inhibition.
Since the mTOR inhibitor temsirolimus (Torisel or CCI-779) has efficacy in RCC 
and promotes autophagy 
, we assessed the functional consequences of coordinate autophagy inhibition with CQ in human RCC cell lines. We found that mTOR inhibition induced selective autophagy of mitochondria (mitophagy) and eliminated RIPKs, the master regulators of necroptosis. Addition of CQ promoted cell death by preventing autophagy, causing RIPK- and ROS-mediated necroptosis. As mTOR inhibition blocked Nrf2 nuclear translocation and antioxidant defense by activating GSK3β, RCC cell lines were sensitized to inhibition of autophagy with CQ and the subsequent ROS production. Thus, autophagy is a cancer therapy survival mechanism to RIPK-dependent necroptosis in the setting of cancer therapy. These findings support clinical assessment of autophagy inhibition to augment the activity of mTOR inhibitors in RCC where Nrf2, autophagy, and RIPK elimination are required for survival.