The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ∼30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.United States. National Institutes of Health (R01CA103866)United States. National Institutes of Health (AI47389)United States. Department of Energy (W81XWH-07-0448)United States. National Institutes of Health (F31 CA180271)United States. National Institutes of Health (F31 CA189437

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Lysosomal nutrients and the mTORC1 pathway

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from student-submitted PDF version of thesis. "February 2019."Includes bibliographical references.The lysosome is the major catabolic organelle, is the site of activation of the master growth regulator mTORC1 (mechanistic target of rapamycin (mTOR) complex 1), and is often deregulated in common diseases, such as cancer. Given the critical role of lysosomes in maintaining cellular homeostasis, a better understanding of lysosomal function and metabolism and its relation to the mTOR pathway is necessary. Most components of the nutrient-sensing machinery upstream of mTORC1 localize to the lysosomal surface, and amino acids generated by lysosomes regulate mTORC1 by promoting its translocation there, a key step in its activation. Activation of mTORC1 by the amino acid arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. To study SLC38A9 function at the lysosome, we developed a novel method for the rapid isolation of intact mammalian lysosomes suitable for metabolite profiling.First, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover a central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes to be used in growth-promoting processes. Pancreatic cancer cells, which use lysosomal protein degradation as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine acts a lysosomal messenger to connect mTORC1 activation and the release of the essential amino acids to drive cell growth. Finally, by performing quantitative proteomic analyses of rapidly isolated lysosomes, we find that ribosome degradation provides the lysosomal arginine that promotes SLC38A9 activation. Lysosome degradation of ribosomes is mediated by NUFIP1 (nuclear fragile X mental retardation-interacting protein 1).The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. Thus, the NUFIP1-mediated degradation of ribosomes provides both the necessary substrate to activate SLC38A9 and the nutrients needed to promote cell survival under starvation. Altogether, this work provides insight into the regulation of lysosomal nutrients and their role in cellular growth and survival.by Gregory A. Wyant.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biolog

Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes

The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H+–adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux

mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient

The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. SLC38A9 is an arginine-regulated transporter of major amino acids, including leucine, providing insights into the regulation of the mTORC pathway and its nutrient-sensing function. Keywords: amino acid sensing; nutrient sensing; mTOR; lysosome; micropinocytosis; autophagyNational Institutes of Health (U.S.) (Grant R01CA103866)National Institutes of Health (U.S.) (Grant R01 CA129105)National Institutes of Health (U.S.) (Grant R37AI47389)United States. Department of Defense (Grant W81XWH-15-1-0230)National Institutes of Health (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Grant F30-CA189333)National Institutes of Health (U.S.) (Grant F32CA210421)National Cancer Institute (U.S.) (Grant R01CA168653)National Cancer Institute (U.S.) (Grant P30CA1405141

Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase–dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid–sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.National Institutes of Health (U.S.) (Grant R01 CA103866)National Institutes of Health (U.S.) (Grant AI47389)United States. Dept. of Defense (W81XWH-07-0448)National Institutes of Health (U.S.) (Fellowship F30CA180754)National Institutes of Health (U.S.) (Fellowship T32 GM007753)National Institutes of Health (U.S.) (Fellowship F31 AG044064)National Institutes of Health (U.S.) (Fellowship F31CA180271)United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship)National Science Foundation (U.S.). Graduate Research Fellowship ProgramAmerican Cancer Society (Ellison Medical Foundation. Postdoctoral Fellowship PF-13-356-01-TBE)Howard Hughes Medical Institut