mTOR and cancer: many loops in one pathway

The mammalian target of rapamycin (mTOR) is a master regulator of cell growth and division that responds to a variety of stimuli, including nutrient, energy, and growth factors. In the last years, a significant number of pieces have been added to the puzzle of how mTOR coordinates and executes its functions. Extensive research on mTOR has also uncovered a complex network of regulatory loops that impact the therapeutic approaches aimed at targeting mTOR.Howard Hughes Medical InstituteNational Institutes of Health (U.S.)Human Frontier Science Program (Strasbourg, France

The metabolic plasticity of B cells.

The humoral response requires rapid growth, biosynthetic capacity, proliferation and differentiation of B cells. These processes involve profound B-cell phenotypic transitions that are coupled to drastic changes in metabolism so as to meet the extremely different energetic requirements as B cells switch from resting to an activated, highly proliferative state and to plasma or memory cell fates. Thus, B cells execute a multi-step, energetically dynamic process of profound metabolic rewiring from low ATP production to transient and large increments of energy expenditure that depend on high uptake and consumption of glucose and fatty acids. Such metabolic plasticity is under tight transcriptional and post-transcriptional regulation. Alterations in B-cell metabolism driven by genetic mutations or by extrinsic insults impair B-cell functions and differentiation and may underlie the anomalous behavior of pathological B cells. Herein, we review molecular switches that control B-cell metabolism and fuel utilization, as well as the emerging awareness of the impact of dynamic metabolic adaptations of B cells throughout the different phases of the humoral response.AE lab is supported by the Retos Projects Program of the Spanish Ministry of Science, Innovation and Universities, the Spanish State Research Agency (AEI/10.13039/501100011033) co-funded by the European Regional Development Fund (PID 2019-104012RB-I00), a FERO Grant for Research in Oncology, and La Caixa Foundation (HR21-00046). AE is an EMBO Young Investigator. YV-G is a recipient of a CNIO Friends Fellowship supported by the Fundacion Domingo Martinez.S

Nutrient-sensing mechanisms and pathways

The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids, lipids and surrogate metabolites are integrated and coordinated at the organismal level through hormonal signals. During food abundance, nutrient-sensing pathways engage anabolism and storage, whereas scarcity triggers homeostatic mechanisms, such as the mobilization of internal stores through autophagy. Nutrient-sensing pathways are commonly deregulated in human metabolic diseases.National Institutes of Health (U.S.) (Grant R01 CA129105)National Institutes of Health (U.S.) (Grant R01 CA103866)National Institutes of Health (U.S.) (Grant R01 AI047389)National Institutes of Health (U.S.) (Grant R21 AG042876)American Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT (Frontier Research Program)Ellison Medical FoundationCharles A. King TrustAmerican Cancer Society (Ellison Medical Foundation Postdoctoral Fellowship PF-13-356-01-TBE

Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma

Follicular lymphoma is an incurable B cell malignancy characterized by the t(14;18) translocation and mutations affecting the epigenome. Although frequent gene mutations in key signaling pathways, including JAK-STAT, NOTCH and NF-κB, have also been defined, the spectrum of these mutations typically overlaps with that in the closely related diffuse large B cell lymphoma (DLBCL). Using a combination of discovery exome and extended targeted sequencing, we identified recurrent somatic mutations in RRAGC uniquely enriched in patients with follicular lymphoma (17%). More than half of the mutations preferentially co-occurred with mutations in ATP6V1B2 and ATP6AP1, which encode components of the vacuolar H+-ATP ATPase (V-ATPase) known to be necessary for amino acid−induced activation of mTORC1. The RagC variants increased raptor binding while rendering mTORC1 signaling resistant to amino acid deprivation. The activating nature of the RRAGC mutations, their existence in the dominant clone and their stability during disease progression support their potential as an excellent candidate for therapeutic targeting.Experimental Cancer Medicine Centre

RagA, but Not RagB, Is Essential for Embryonic Development and Adult Mice

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cues from growth factors and nutrients to control metabolism. In contrast to the growth factor input, genetic disruption of nutrient-dependent activation of mTORC1 in mammals remains unexplored. We engineered mice lacking RagA and RagB genes, which encode the GTPases responsible for mTORC1 activation by nutrients. RagB has limited expression, and its loss shows no effects on mammalian physiology. RagA deficiency leads to E10.5 embryonic death, loss of mTORC1 activity, and severe growth defects. Primary cells derived from these mice exhibit no regulation of mTORC1 by nutrients and maintain high sensitivity to growth factors. Deletion of RagA in adult mice is lethal. Upon RagA loss, a myeloid population expands in peripheral tissues. RagA-specific deletion in liver increases cellular responses to growth factors. These results show the essentiality of nutrient sensing for mTORC1 activity in mice and its suppression of PI3K/Akt signaling.United States. National Institutes of Health (R01 CA129105)United States. National Institutes of Health (R01 CA103866)United States. National Institutes of Health (R01 AI047389)United States. National Institutes of Health (R21 AG042876)American Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT. Frontier Research ProgramEllison Medical FoundationUnited States. National Institutes of Health (AG041765)National Cancer Institute (U.S.) (F31CA167872

mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase

The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that is stimulated by amino acids. Amino acids activate the Rag guanosine triphosphatases (GTPases), which promote the translocation of mTORC1 to the lysosomal surface, the site of mTORC1 activation. We found that the vacuolar H+–adenosine triphosphatase ATPase (v-ATPase) is necessary for amino acids to activate mTORC1. The v-ATPase engages in extensive amino acid–sensitive interactions with the Ragulator, a scaffolding complex that anchors the Rag GTPases to the lysosome. In a cell-free system, ATP hydrolysis by the v-ATPase was necessary for amino acids to regulate the v-ATPase-Ragulator interaction and promote mTORC1 translocation. Results obtained in vitro and in human cells suggest that amino acid signaling begins within the lysosomal lumen. These results identify the v-ATPase as a component of the mTOR pathway and delineate a lysosome-associated machinery for amino acid sensing.Damon Runyon Cancer Research FoundationMillennium Pharmaceuticals, Inc.American Lebanese Syrian Associated CharitiesHoward Hughes Medical Institut

Bias in Intracellular Luminescence Thermometry: The Case of the Green Fluorescent Protein

Measurement of intracellular temperature in a fast, accurate, reliable, and remote manner is crucial for the understanding of cellular processes. Nanothermometers based on the green fluorescence protein (GFP) are of special interest because intracellular temperature readouts can be obtained from the analysis of the polarization state of its luminescence. Despite the good results provided by GFP thermometers, the reliability of their intracellular thermal readouts is still a question of debate. Here, light is shed on this issue by introducing cell activity as a relevant bias mechanism that prevents the use of GFP for reliable intranuclear thermal measurements. Experimental evidence that this lack of reliability can affect not only GFP but also other widely used thermometers such as semiconductor nanocrystals is provided. It is discussed how differences observed between calibration curves obtained in presence and absence of cell activity can inform about the presence of bias. The presented results and discussion are aimed to warn the community working in intracellular thermometry and encourage authors to approach the issue in a conscious manner. The performance and reliability of the chosen intracellular thermometers must be judiciously assessed. This is the only way intracellular thermometry can progress and deliver indisputable resultsThis work was financed by the Spanish Ministerio de Innovación y Ciencias under Project Nos. RTI2018-101050-J-I00, NANONERV PID2019- 106211RB-I00, and EIN2020-112419. Additional funding was provided by the European Union Horizon 2020 FETOpen project NanoTBTech (Grant No. 801305). P.R.-S. is grateful for a Juan de la Cierva – Incorporación scholarship (Grant No. IJC2019-041915-I). A.E. is grateful to Retos Projects Program of the Spanish Ministry of Science, Innovation, and Universities, the Spanish State Research Agency, co-funded by the European Regional Development Fund (A.E. is an EMBO Young Investigator). S.T. is grateful to AECC (Spanish Association Against Cancer) IDEAS21989THOM

Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst

Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection