Abstract Lung adenocarcinoma (LUAD) is a multifaceted and genetically diverse cancer, shaped by a myriad of oncogenic and tumor suppressive events. Through recent clinical and in vitro investigations, we have discovered that the TBX2 subfamily of genes, encompassing TBX2, TBX3, TBX4, and TBX5, are linked to LUAD progression. Specifically, we have observed marked downregulation of their expression in clinical LUAD specimens, and reduced growth in LUAD cell lines when the subfamily is re-activated- hinting at a potential tumor suppressive function. Paradoxically, the role of TBX2 subfamily genes in other cancer types remains enigmatic, with varying reports of both oncogenic and tumor suppressive effects. In this study, we harnessed the power of Tumor Barcoding with Ultradeep Barcode Sequencing technology (Tuba-seq) and CRISPR/Cas9-mediated genome editing to systematically investigate the effects of knocking-out Tbx genes and associated partners EGR1, CHD2, A20 and ATF3 in LUAD preclinical models. We investigated their effects in both normal lung epithelium and Ras-driven lung tumors via in situ gene editing and assessed tumor burden via histology and targeted DNA sequencing of tumor barcodes. TBX2 subfamily gene losses appear to have a profound effect on tumor initiation and early growth, which somewhat attenuates with progression. In Ras-driven tumors, we found that these genes are moderately tumor suppressive when measuring mean growth rate, although these effects (sans Egr1 loss) are more pronounced at 6 weeks of growth than at 20 weeks. Strikingly, however, we observed an unprecedented increase in total tumor burden when deleting these genes in genomically-normal (Kras w.t.) cells, including 4-20+ fold increase in Tbx2, Tbx3, Tbx4, and Tnfaip3-deficient cell lineages 20-week following transduction. Increases in total lineage burden of this magnitude are typically only seen for frequently-deleted hallmark tumor suppressor genes such as Tp53, Stk11, or Pten. This increased burden, however, seems to primarily affect early-stage progression, as no appreciable increases in mean lineage size relative to w.t. lineages are observed after 40 weeks of growth. Our study reveals the context-specific functions of the TBX2 subfamily while introducing the first in vivo model for TBX2-driven LUADs. This model facilitates the development of targeted therapeutic strategies and encourages accurate tumor modeling across various progression stages and diverse genetic backgrounds. Citation Format: Athar Khalil, Jaff Maltas, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher McFarland. Tuba-seq and CRISPR/Cas9 analysis of TBX2 subfamily genes: Exploring contextual tumor suppression in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 634.

Lung adenocarcinoma (LUAD) is a complex cancer driven by diverse combinations of oncogenic and tumor suppressive events. The TBX2 subfamily genes, including TBX2, TBX3, TBX4, and TBX5, are known to play a crucial role in lung development as master transcriptional regulators. Our previous clinical and in vitro studies found that their expression is significantly suppressed in LUADs, suggesting a potential tumor suppressor role through modulating tumor methylation patterns in early tumorigenesis. However, the role of TBX2 subfamily genes in other cancer types is paradoxical, with both oncogenic and tumor suppressive effects being reported. Herein, we employed the Tumor Barcoding with Ultradeep Sequencing (Tuba-seq) and CRISPR/Cas9-mediated genome editing technologies to investigate the putative tumor suppressive role of TBX2 genes functionally and quantitatively and their downstream targets in genetically engineered mouse models of LUAD. Lung tumors were initiated by intratracheal intubation with pooled lentiviral-Cre vectors targeting each gene independently with two different sgRNAs per gene. To enable the unique identification of each tumor and its corresponding sgRNA, we utilized Lenti-sgRNA/Cre vectors harboring barcodes specific to each targeted gene. Initial assessment of tumor burden was carried out through fluorescence microscopy, lung weight measurements, and histological analysis. To simultaneously determine the number of neoplastic cells corresponding to each gene knockout, the barcoded region was amplified from genomic DNA extracted from bulk tumor-bearing lung samples and sequenced. Quantification of tumor sizes was carried at 6 and 20 weeks after tumor initiation. Hyperplasia, adenomas, and/or early adenocarcinomas within the lungs were detected among different genetic backgrounds and throughout the study time intervals. Specifically, the inactivation of Tbx2, Tbx3, Tbx4, Tbx5, and Cdh2, increased tumor initiation and growth in the oncogene-negative mouse model (n=8). However, strikingly, inactivation of Tbx3, Tbx4, and Cdh2 suppressed tumorigenesis in a KRAS-driven genetic background while Tbx2, Tbx5, and Tnfaip3, consistently exhibited tumor suppressive effects (n=9). Further investigation is needed to understand these newly discovered interactions between the TBX2 subfamily critical developmental pathway and Ras signaling in modulating lung cancer progression. These insights emphasize the importance of considering tumor suppressor phenotypic heterogeneity and their context-dependent roles when developing targeted therapeutic approaches for lung adenocarcinoma and other cancers. Citation Format: Athar Khalil, Mira Rahm, Zachary Faber, Madeline Bedrock, Xiangzhen Wei, Bindi Patel, Christopher Mcfarland. In vivo TuBa-seq growth profiling identifies a differential role of the Tbx2 subfamily in oncogene-negative versus Kras-driven lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A177.

Introduction: T cell activation is critical in the initiation and potentiation of anti-tumor immune responses. Hematopoietic Progenitor Kinase 1 (HPK1/MAP4K1) is a member of the MAP4K family whose activity has been demonstrated to restrain T cell activation through phosphorylation of SLP-76 at Serine 376 leading to TCR disassembly. Mediators generated in the tumor microenvironment (TME) such as adenosine, PGE2 and TGFβ can dampen T cell activity and present a significant barrier to cancer therapy. HPK1 inhibition may allow for enhanced T cell activation under such suppressive conditions. Herein, we describe studies to assess the effects of HPK1 inhibition on T cell activation and in alleviating suppression. We also test the ability of HPK1 inhibition in combination with adenosine receptor antagonism to further amplify T cell activation. Methods: CD8+ T cells were isolated from human blood and RNA was isolated for qPCR analysis of MAP4K1-7 expression. Cells were activated using CD3/28 stimulation and supernatants were assayed for IL-2, IFNɣ and TNFα using CBA. CRISPR knockout of HPK1 in CD8+ T cells and reference or in-house HPK1 inhibitors used to characterize HPK1 function. We used a flow cytometry-based assay to quantify activation markers CD69 and CD25, and phospho-SLP-76 in isolated CD8+ T cells and in human and mouse whole blood. Results: Using publicly available gene expression databases, we identified that HPK1 is primarily expressed in immune cells, with highest expression in T cells, B cells, and dendritic cells. In-house gene expression analyses demonstrated that HPK1 is the predominant MAP4K family member in human T cells. CRISPR knockout in primary human CD8+ T cells was used to demonstrate that HPK1 but not MAP4K3 and MAP4K4 negatively regulates T cell activation. Consistent with this, reference and in-house HPK1 kinase inhibitors reduced phosphorylation of SLP-76 in both isolated T cells and human and mouse whole blood. In human T cells, HPK1 inhibition dose-dependently increased IL-2, IFNɣ and TNFα secretion, highlighting the negative role of HPK1 in T cell activation. Using adenosine, PGE2 and TGFβ, expression of CD69 and T cell cytokine secretion is diminished. HPK1 inhibition restored T cell activation to unsuppressed levels. Consistent with these results, HPK1KO CD8+ T cells displayed significant resistance to adenosine, PGE2 and TGFβ-induced immunosuppression. Finally, upon suppression using adenosine, combining HPK1 inhibition with adenosine receptor antagonism further elevated T cell activation above individual inhibitor treatments alone. Conclusion: These data demonstrate that HPK1 plays a significant role in dampening T cell activation and provide a strong therapeutic rationale for targeting HPK1 to relieve T cell immunosuppression in the TME and amplify anti-tumor immune responses. Citation Format: Rajesh K. Singh, Rameshwari Rayaji, Bindi Patel, Kristen Zhang, Sachie Marubayashi, Soonweng Cho, Stefan Garrido-Shaqfeh, Joseph Kulusich, Cesar Meleza, Nidhi Tibrewal, Joice Thomas, Pradeep Nareddy, Ehesan Sharif, Sharon Zhao, David Green, Manmohan R. Leleti, Jay P. Powers, Daniel DiRenzo, Matthew J. Walters. HPK1 inhibition enhances T cell activation and relieves the immunosuppressive phenotype of inhibitory signals found in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1367.

INTRODUCTION: Tumors employ many strategies to attenuate immune responses. High levels of extracellular adenosine generated in the tumor microenvironment engage A2a and A2b adenosine receptors on immune cells, resulting in immunosuppression. Expression of A2aR and A2bR can vary by cell type, with T cells predominantly expressing A2aR while myeloid cells express both A2aR and A2bR. We have previously shown that etrumadenant, a dual A2aR/A2bR antagonist, blocks the immunosuppressive effects of adenosine in immune cells and enhances anti-tumor immune responses in mouse syngeneic tumors. Using dual and selective adenosine receptor antagonists, we assessed the contribution of these receptors to adenosine-mediated phenotypes in immune and cancer cells. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using CD3/CD28/CD2 stimulation and cytokines were analyzed by cytokine bead array at 72 hours. Primary human dendritic cells (DC) were isolated from healthy blood and matured with LPS/IFN-γ for 24 hours. Cancer cell lines were purchased from ATCC. The adenosine analogue NECA was used to stimulate A2aR/A2bR-mediated signaling. RESULTS: Activated human CD8+ T cells stimulated in the presence of NECA showed suppression of activation markers (CD69) and cytokine production (IFN-γ, IL-2 and granzyme B). As expected, we observed similar rescue of this phenotype with both etrumadenant and an A2aR-specific antagonist owing to the sole expression of A2aR on T cells. In contrast, primary DC have comparable expression of A2aR and A2bR, suggesting that dual blockade may provide greater resistance to adenosine-mediated suppression than A2aR antagonism. Indeed, etrumadenant was able to attenuate the adenosine-mediated upregulation of IL-10 and enhance IL-12p70 production, whereas a comparable A2aR-specific antagonist showed no significant rescue versus NECA-stimulated controls. These observations may be extended to suppressive myeloid populations as well as tumor-resident macrophages and myeloid-derived suppressor cells isolated from mouse syngeneic tumors, which have very high expression of both A2aR and A2bR. RNA-sequencing identified a cassette of genes regulated by adenosine-signaling in these cells, which were largely reversed by etrumadenant. Finally, cultured human cancer cell lines have high expression of A2bR, which has been implicated in driving their tumorigenesis. Etrumadenant reversed adenosine-stimulated gene expression changes in non-small cell lung cancer cell lines, restoring enriched pathways driven by adenosine signaling. CONCLUSIONS: Taken together, these results show an important role for A2aR/A2bR in adenosine-mediated immunosuppression and provide a mechanistic rationale for stimulation of anti-tumor immune responses with the dual adenosine receptor antagonist etrumadenant. Citation Format: Sachie Marubayashi, Bindi Patel, Livia Yamashiro, Dana Piovesan, Sean Cho, Jenna Jeffrey, Manmohan Leleti, Jay Powers, Matt Walters, Daniel DiRenzo. Dual A2aR/A2bR antagonism with etrumadenant (AB928) eliminates the suppressive effects of adenosine on immune and cancer cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 256.

Background T cell activation is critical in the initiation and potentiation of anti-tumor immune responses. Hematopoietic Progenitor Kinase 1 (HPK1) is a member of the MAP4K family whose activity restrains T cell activation through phosphorylation of SLP-76 (pSLP-76) at Serine 376, leading to disassembly of the TCR complex. Mouse genetic deletion and kinase dead mutants of HPK1 have been shown to enhance T cell activity and combine with immune checkpoint inhibition. Therefore, we sought to demonstrate that inhibitors of HPK1 activity can increase T cell activation, drive antigen-specific cancer cell killing, and combine with immune checkpoint blockade to amplify anti-tumor T cell responses. Herein, we describe the characterization of novel inhibitors of HPK1 and assess their effects on T cell activation in combination with PD-1 blockade. Methods Measurement of pSLP-76 using a flow cytometry-based assay was employed to assess HPK1 activity in human and mouse whole blood. Jurkat cells, human CD8+ T cells and PBMCs were activated using anti-CD3/CD28 stimulation, CEF peptide pools, or Staphylococcal enterotoxin A (superantigen), in the presence of HPK1 inhibitors; levels of IL-2, IFN-γ and TNF-α were assayed in supernatants by cytokine bead array. Mouse OT-1 splenocytes were stimulated with SIINFEKL peptide and co-cultured with E.G7-OVA cells to assess cytokine production and target cell killing. Results HPK1 inhibitors exhibited a potent, concentration-dependent reduction in pSLP-76, with a concurrent increase in IL-2 secretion in both Jurkat and human CD8+ T cells. Human T cells also demonstrated increased IFN-γ and TNF-α secretion as well as a greater percentage of activated CD69+ cells with HPK1 inhibition. These increases in T cell activation were mirrored in antigen-specific OT-1 T cell assays, in which HPK1 inhibition enhanced IFN-γ production in response to OVA peptide and greater killing of E.G7-OVA cancer cells. Similarly, antigen-recall assays demonstrated that inhibition of HPK1 increased IFN-γ production from human PBMC stimulated with CEF peptide and combined with PD-1 blockade to further enhance cytokine production. Consistent with these results, HPK1 inhibition and PD-1 blockade increased cytokine secretion in superantigen-stimulated human PBMC individually and further enhanced cytokine production in combination. Taken together, these data demonstrate that the combined activity of HPK1 inhibition with checkpoint therapy may yield greater anti-tumor T cell activity. Conclusions These data demonstrate that pharmacological inhibition of HPK1 amplifies antigen-specific T cell activation alone or in combination with immune checkpoint blockade and provide a strong mechanistic rationale for targeting HPK1 to amplify anti-tumor immune responses.

Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration.

Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression.

Objective: This case demonstrates the variability in presentation of CNS Lyme. Background: Spirochetal infections have a broad spectrum of clinical manifestations and severity, with 10–15% of those infected with Lyme disease having CNS involvement. This is due to meningeal seeding of the spirochetes in early disseminated Lyme disease. Due to variability in presentation, diagnosis is often difficult to make. Design/Methods: NA Results: A 50-year-old previously healthy man presented to our hospital with acute onset total right sided facial tingling and weakness. Prior to hospitalization, patient had presented to his primary doctor three weeks prior for painful paresthesias of his low back, right hip, right buttocks, and left thigh. His primary doctor ordered a hip XR, which was negative, and prescribed Flexeril. Due to persistent pain and paresthesias, patient was then started on prednisone, and he then developed a right facial droop, pressure-like headache, and double vision leading to his presentation to the hospital. At our initial assessment, patient had a right 7th nerve palsy with House-Brackmann grade of IV, right 5th nerve palsy, and right 6th nerve palsy. Motor and sensory testing elsewhere were normal, although patient had non-specific complaints of shoulder and hip girdle pain with paresthesias into his extremities. Lumbar puncture was performed, which was significant for CSF pleocytosis and eventually CNS Lyme returned as positive. Patient empirically started on IV Ceftriaxone after lumbar puncture and had eventual clinical improvement. Conclusions: This case illustrates the prolonged time to diagnosis, and possible worsening with steroidal treatment, in a patient with CNS Lyme disease. While our patient’s initial presenting symptoms appear non-specific, the initial complaint concerning for mechanical radiculopathy without apparent mechanical precipitant could suggest the possibility of a radiculoneuritis. The constellation of radiculoneuritis, multiple cranial neuropathies, and CSF pleocytosis helped us to make the diagnosis of CNS Lyme. Disclosure: Dr. Patel has nothing to disclose.

Homeostatic control of core body temperature is essential for survival. Temperature is sensed by specific neurons, in turn eliciting both behavioral (i.e., locomotion) and physiologic (i.e., thermogenesis, vasodilatation) responses. Here, we report that a population of GABAergic (Vgat-expressing) neurons in the dorsolateral portion of the dorsal raphe nucleus (DRN), hereafter DRNVgat neurons, are activated by ambient heat and bidirectionally regulate energy expenditure through changes in both thermogenesis and locomotion. We find that DRNVgat neurons innervate brown fat via a descending projection to the raphe pallidus (RPa). These neurons also densely innervate ascending targets implicated in the central regulation of energy expenditure, including the hypothalamus and extended amygdala. Optogenetic stimulation of different projection targets reveals that DRNVgat neurons are capable of regulating thermogenesis through both a "direct" descending pathway through the RPa and multiple "indirect" ascending pathways. This work establishes a key regulatory role for DRNVgat neurons in controlling energy expenditure.

Obesity is associated with changes in the immune system that significantly hinder its ability to mount efficient immune responses. Previous studies have reported a dysregulation of immune responses caused by lipid challenge; however, the mechanisms underlying that dysregulation are still not completely understood. Autophagy is an essential catabolic process through which cellular components are degraded by the lysosomal machinery. In T cells, autophagy is an actively regulated process necessary to sustain homeostasis and activation. Here, we report that CD4+ T cell responses are inhibited when cells are challenged with increasing concentrations of fatty acids. Furthermore, analysis of T cells from diet-induced obese mice confirms that high lipid load inhibits activation-induced responses in T cells. We have found that autophagy is inhibited in CD4+ T cells exposed in vitro or in vivo to lipid stress, which causes decreased autophagosome formation and degradation. Supporting that inhibition of autophagy caused by high lipid load is a key mechanism that accounts for the effects on T cell function of lipid stress, we found that ATG7 (autophagy-related 7)-deficient T cells, unable to activate autophagy, did not show additional inhibitory effects on their responses to activation when subjected to lipid challenge. Our results indicate, thus, that increased lipid load can dysregulate autophagy and cause defective T cell responses, and suggest that inhibition of autophagy may underlie some of the characteristic obesity-associated defects in the T cell compartment.Abbreviations: ACTB: actin, beta; ATG: autophagy-related; CDKN1B: cyclin-dependent kinase inhibitor 1B; HFD: high-fat diet; IFNG: interferon gamma; IL: interleukin; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MAPK8/JNK: mitogen-activated protein kinase 8; LC3-I: non-conjugated form of MAP1LC3B; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3B; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; NFATC2: nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2; NLRP3: NLR family, pyrin domain containing 3; OA: oleic acid; PI: propidium iodide; ROS: reactive oxygen species; STAT5A: signal transducer and activator of transcription 5A; TCR: T cell receptor; TH1: T helper cell type 1.

Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule-based minus-end-directed motor proteins, the well-characterized dynein, and the less-studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti-aging purposes.

3,7-Dihydroxytropolones (3,7-dHTs) are highly oxygenated troponoids that have been identified as lead compounds for several human diseases. To date, structure-function studies on these molecules have been limited due to a scarcity of synthetic methods for their preparation. New synthetic strategies towards structurally novel 3,7-dHTs would be valuable in further studying their therapeutic potential. Here we describe the successful adaptation of a [5 + 2] oxidopyrilium cycloaddition/ring-opening for 3,7-dHT synthesis, which we apply in the synthesis of a plausible biosynthetic intermediate to the natural products puberulic and puberulonic acid. We have also tested these new compounds in several biological assays related to human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV) in order to gain insight into structure-functional analysis related to antiviral troponoid development.

Ca2+ signals were reported to control lipid homeostasis, but the Ca2+ channels and pathways involved are largely unknown. Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca2+ channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and peroxisome proliferator-activated receptor α (PPARα). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. Our data provide evidence for an important role of SOCE in lipid metabolism.

Hunger, driven by negative energy balance, elicits the search for and consumption of food. While this response is in part mediated by neurons in the hypothalamus, the role of specific cell types in other brain regions is less well defined. Here, we show that neurons in the dorsal raphe nucleus, expressing vesicular transporters for GABA or glutamate (hereafter, DRN Vgat and DRN VGLUT3 neurons), are reciprocally activated by changes in energy balance and that modulating their activity has opposite effects on feeding-DRN Vgat neurons increase, whereas DRN VGLUT3 neurons suppress, food intake. Furthermore, modulation of these neurons in obese (ob/ob) mice suppresses food intake and body weight and normalizes locomotor activity. Finally, using molecular profiling, we identify druggable targets in these neurons and show that local infusion of agonists for specific receptors on these neurons has potent effects on feeding. These data establish the DRN as an important node controlling energy balance.

A role for Ca signaling in the regulation of lipid metabolism has been suggested, but the Ca channels involved in this process remain elusive. Store-operated Ca entry (SOCE) is a ubiquitous Ca influx pathway that is regulated by the endoplasmic reticulum (ER) Ca sensors stromal interaction molecule (STIM) 1 and 2, and the plasma membrane Ca channel ORAI1. SOCE-deficient mice with abolished SOCE accumulate pathological amounts of lipid droplets in the liver, heart and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 and ORAI1 show lipid droplet accumulation, suggesting that the role of SOCE in lipid metabolism is conserved in humans. Functionally, SOCE contributes to regulating levels and usage of intracellular lipids by modulating lipolysis, lipophagy and mitochondrial function. Mechanistically, SOCE controls expression of PGC-1 and PPAR, master regulators of lipid metabolism. Our data demonstrate that SOCE is a crucial regulator of lipid metabolism.