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Additional file 1: Figure S1. Components of the nutrient-mTORC1 pathway. Cartoon with the regulatory network of genes within the mTORC1 pathway. Figure S2 (related to main Fig. 1). Genetic associations. Figure S3 (related to main Fig. 3). Transcriptomic consequences of rs2291007. Figure S4 (related to main Fig. 4). A Fnip2C Knock-in mouse to model FNIP2 rs2291007. Figure S5 (related to main Fig. 5). Impact of the engineered T-to-C substitution on mouse adiposity. Figure S6. Un-cropped Western blots.

Additional file 3. Review history.

16 p.-4 fig.-1 graph. abst., Temperature measurement at the nano-scale has brought insight to a wide arrayof research interests in modern chemis-try, physics, and biology. These measure-ments have been enabled by the adventof nanothermometers, which relay nano-scale temperature information throughthe analysis of their intrinsic photo-physical behavior. In the past decade, several nanothermometers have beendeveloped including dyes, nanodiamonds, fluorescent proteins, nucleotides,and nanoparticles. However, temperature measurement using intact DNA hasnot yet been achieved. Here, we present a method to study the temperaturesensitivity of the DNA molecule within a physiologic temperature range whencomplexed with fluorescent dye. We theoretically and experimentally reportthe temperature sensitivity of the DNA-Hoechst 33342 complex in differentsizes of double-stranded oligonucleotides and plasmids, showing its potentialuse as a nanothermometer. These findings allow for extending the thermalstudy of DNA to several research fields including DNA nanotechnology, opti-cal tweezers, and DNA nanoparticles., This work was supported by RETOS RTI2018-101050-J-I00 (ST), National Institute of General Medical Sciences of the National Institutes of Health under award numberR01GM124133 (AA), Ramon y Cajal Grant (RYC-2013-13546) and by SAF2015-67538-R (MCIU/AEI/FEDER, UE) (AE)

SUMMARY B lymphocytes are exquisitely sensitive to fluctuations in nutrient signaling by the Rag GTPases, and 15% of follicular lymphomas (FLs) harbor activating mutations in RRAGC. Hence, a potential therapeutic approach against malignant B cells is to inhibit Rag GTPase signaling, but because such inhibitors are still to be developed, efficacy and safety remain unknown. We generated knockin mice expressing a hypomorphic variant of RagC (Q119L); RagCQ119L/+ mice are viable and show attenuated nutrient signaling. B lymphocyte activation is cell-intrinsically impaired in RagCQ119L/+ mice, which also show significant suppression of genetically induced lymphomagenesis and autoimmunity. Surprisingly, no overt systemic trade-offs or phenotypic alterations caused by partial suppression of nutrient signaling are seen in other organs, and RagCQ119L/+ mice show normal longevity and normal age-dependent health decline. These results support the efficacy and safety of moderate inhibition of nutrient signaling against pathological B cells., Graphical abstract, In brief By generating knockin mice expressing a hypomorphic variant of the RagC GTPase, Ortega-Molina et al. show that partial inhibition of nutrient signaling may be a safe and efficacious approach against pathogenic B cells and B cell lymphomas.

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity. General: We thank Prof. Jose Maria. Ordovas for his kind suggestions; nutritionists Helena Marcos-Pasero, Elena Aguilar-Aguilar and Isabel Espinosa-Salinas for their help with volunteers management; Rosa Serrano for her help with animal experiments; Susana Molina for her advice with PBMC isolation; Luisa Mariscal, Domingo Fernandez, Lola Martinez, Diego Megias, Patricia Gonzalez, Fernando Pelaez, Anabel Sanz, Carolina Pola, Celia de la Calle, Ana Ortega, Ana Sagrera, Jose Miguel Frade, Elena Lopez-Guadamillas, Maribel Munoz, Susana Llanos, Andres Fernandez, Aranzazu Sierra, Andres Lopez, Noemi Haro and Ildefonso Rodriguez for their excellent technical and scientific support. Work at the laboratory of P.J.F.M. is funded by the Ramon Areces Foundation, (CIVP18A3891), Asociacion Espanola contra el Cancer-AECC (SIRTBIO-LABAE18008FERN), a Ramon y Cajal Award from the Spanish Ministry of Science, Innovation and Universities (MICINN) (RYC-2017-22335), RETOS projects Program of MICINN (SAF2017-85766-R) and the Portuguese Foundation for Science and Technology (FCT-MCTES, SFRH/BD/124022/2016). Work at the laboratory of ARM was funded by the MICINN (PID2019-110183RB-C21), Regional Government of Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM) and the Ramon Areces Foundation. Work at the laboratory of A.D.R. Funded by the Comunidad de Madrid-Talento Grant 2018-T1/BMD-11966 and the MICINN PID-2019-106893RA-100. Work at the laboratory of L.D. is funded by projects from the Health Research Fund (ISCIII FIS PI14/01374 and FISPI17/00508) and from a Manuel de Oya research fellowship from the Beer and Health Foundation. Work at the laboratory of A.E. is funded by a Ramon y Cajal Award from MICINN (RYC-2013-13546) and RETOS projects Program of the MICINN, co-funded by the European Regional Development Fund (ERDF) (SAF2015-67538-R). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and the "laCaixa" Foundation. Sí

Cyclin D3 drives a unique form of “inertial” cell cycling responsible for clonal expansion of B cells in the germinal center dark zone., During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma–associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation., Graphical Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state., The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and hormonal signals to control metabolism. Here the authors investigate the effects of constitutive nutrient signaling through genetic activation of RagA in adult mice and show that constitutive nutrient signaling regulates the response to feeding-fasting cycles and does not drive liver cancer.

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

During affinity maturation, germinal center (GC) B cells alternate between proliferation and so-matic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively-selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, to clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.

Over the last years, there have been relevant advances in cancer research and in treatment based on specifc genetic alterations improving survival in some tumors. Nevertheless, progress is still dismal in most carcinomas when are disseminated with metastasis; the survival is less than 30% in most cases. Then, we need to move forward looking for new paradigms which may explain the complex mechanisms involved in carcinogenesis and in new treatment approaches., We thank to the University of Zaragoza for the opportunity to organize this meeting and to Telefonica, Ibercaja and the Fero Foundation for its support.

Foxp3+ regulatory T (T reg) cells are pivotal regulators of immune tolerance, with T cell receptor (TCR)-driven activated T reg (aT reg) cells playing a central role; yet how TCR signaling propagates to control aT reg cell responses remains poorly understood. Here we show that TCR signaling induces expression of amino acid transporters, and renders amino acid-induced activation of mTORC1 in aT reg cells. T reg cell-specific ablation of the Rag family small GTPases RagA and RagB impairs amino acid-induced mTORC1 signaling, causing defective amino acid anabolism, reduced T reg cell proliferation, and a rampant autoimmune disorder similar in severity to that triggered by T reg cell-specific TCR deficiency. Notably, T reg cells in peripheral tissues, including tumors, are more sensitive to Rag GTPase-dependent nutrient sensing. Ablation of RagA alone impairs T reg cell accumulation in the tumor, resulting in enhanced antitumor immunity. Thus, nutrient mTORC1 signaling is an essential component of TCR-initiated T reg cell reprogramming, and Rag GTPase activities may be titrated to break tumor immune tolerance., Memorial Sloan-Kettering Cancer Center (Support Grant/Core Grant P30CA08748)

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 Research, Starr Foundation, David H. Koch Institute for Integrative Cancer Research at MIT. Frontier Research Program, Ellison Medical Foundation, United States. National Institutes of Health (AG041765), National Cancer Institute (U.S.) (F31CA167872)

During the humoral response, B cells undergo a sudden anabolic shift that requires high cellular nutrient levels to sustain the subsequent proliferative burst. Follicular lymphoma (FL) originates from B cells that have participated in the humoral response, and 15% of FL samples have selected for point, activating mutations in RRAGC, a member of the Rag GTPase family that controls the activation of the mechanistic target of rapamycin complex 1 downstream of the sensing of cellular nutrients. S74C and T89N, two of the most frequent activating single-amino acid changes in RRAGC, when targeted to the endogenous Rragc locus in mice, confer only a partial insensitivity to nutrient deprivation but strongly exacerbate B-cell responses and accelerate lymphomagenesis. Surprisingly, this moderate increase in nutrient signaling affected the interaction of B cells with the cellular microenvironment, synergizing their effects on mTORC1 activation with paracrine cues from the supportive T-cell microenvironment that activate B cells via the PI3K–Akt–mTORC1 axis. Hence, Rragc mutations sustain induced germinal centers and murine and human FL in the presence of decreased T-cell help. From a therapeutic standpoint, Rragc mutations impose a selective vulnerability to pharmacologic inhibition of mTORC1. Our results support a model in which activating mutations in the nutrient signaling pathway foster lymphomagenesis by corrupting a nutrient-dependent control over paracrine signals from the T-cell microenvironment. While pharmacologic inhibition of mTORC1 with rapamycin yielded exciting preclinical responses in murine lymphomas with activating mutations in Rragc, targeting the nutrient signaling cascade itself, instead of using such allosteric, partial inhibition of mTOR, may constitute a more efficacious intervention. Because nutrient signaling inhibitors are still in development phase, their efficacy and safety remain unproven. Previous genetic approaches to investigate the consequences of inhibition of Rag GTPase signaling relied on deletion of the Rags in mice and led to severe phenotypes and death. Incomplete inhibition of nutrient signaling, an approach that would mirror more closely the effect of small molecules, has not been pursued to support both their efficacy and safety. We have generated knock-in mice endogenously expressing a point-mutant form of RagC (Q119L) that partially suppresses nutrient signaling. RagCQ119L/Q119L mice are not viable, but RagCQ119L/+ mice show minimal phenotypic alterations with partially decreased nutrient signaling. While B-cell development was unaffected, B-cell activation and the humoral response were impaired in RagCQ119L/+ in a B cell-intrinsic manner. When bred to the FL- and autoimmunity-prone strain VavP-Bcl2, RagCQ119L/+ mice were protected against development of both FL and autoimmunity. No obvious systemic tradeoff for the suppression of nutrient signaling seems to occur, because RagCQ119L/+ mice show normal physiology and longevity with a similar age-dependent health decline. Altogether, our work supports the oncogenicity of activating mutations in components of the nutrient signaling pathway, such as RagC, and both the efficacy and safety of a moderate inhibition of nutrient signaling against pathologic B cells without detrimental systemic effects. Citation Format: Ana Ortega-Molina, Cristina Lebrero-Fernández, Nerea Deleyto-Seldas, Alba Sanz, Alejo Efeyan. Oncogenic Rag GTPase signaling links cellular nutrients with the FL microenvironment [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA13.

In the last decade, technological advances in chemistry and photonics have enabled real-time measurement of temperature at the nanoscale. Nanothermometers, the probes specifically designed to relay these nanoscale temperature changes provide a high degree of temperature, temporal, and spatial resolution and precision. Several different approaches have been proposed, including micro-thermocouples, luminescence, and fluorescence polarization anisotropy-based nanothermometers. Anisotropy-based nanothermometers excel in terms of biocompatibility because they can be built from endogenous proteins conjugated to dyes, minimizing any system perturbation. Moreover, the resulting fluorescent proteins can retain their native structure and activity while performing the temperature measurement, allowing precise temperature recordings from the native environment or during an enzymatic reaction in any given experiment system. To facilitate future use of these nanothermometers in research, here we present a theoretical model that predicts the optimal sensitivity for anisotropy-based thermometers starting with any protein or dye, based on protein size and dye fluorescence lifetime. Using this model, most of the proteins and dyes can be converted to nanothermometers. The utilization of these nanothermometers by a broad spectrum of disciplines within the scientific community will bring new knowledge and understanding that today remains unavailable with current techniques.

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 Foundation, Millennium Pharmaceuticals, Inc., American Lebanese Syrian Associated Charities, Howard Hughes Medical Institute

Nature 547, 109–113 (2017); doi:10.1038/nature22964 In this Letter, there are errors in Extended Data Figs 5, 8 and 9, and the affiliation of an author. The affiliations for author Violeta Serra should include number 2 (CIBERONC, Instituto de Salud Carlos III, C/ Monforte de Lemos 3-5, Pabellon 11, Planta 0, 28029 Madrid, Spain).

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation1,2. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model3 and human biopsies4 of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus5 exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation,. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.