Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy.

The heart either hypertrophies or dilates in response to familial mutations in genes encoding sarcomeric proteins, which are responsible for contraction and pumping. These mutations typically alter calcium-dependent tension generation within the sarcomeres, but how this translates into the spectrum of hypertrophic versus dilated cardiomyopathy is unknown. By generating a series of cardiac-specific mouse models that permit the systematic tuning of sarcomeric tension generation and calcium fluxing, we identify a significant relationship between the magnitude of tension developed over time and heart growth. When formulated into a computational model, the integral of myofilament tension development predicts hypertrophic and dilated cardiomyopathies in mice associated with essentially any sarcomeric gene mutations, but also accurately predicts human cardiac phenotypes from data generated in induced-pluripotent-stem-cell-derived myocytes from familial cardiomyopathy patients. This tension-based model also has the potential to inform pharmacologic treatment options in cardiomyopathy patients.

Chronic calcium signaling in IgE+ B cells limits plasma cell differentiation and survival.

In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. To better understand IgE-BCR-mediated control of IgE responses, we developed whole-genome CRISPR screening that enabled comparison of IgE+ and IgG1+ B cell requirements for proliferation, survival, and differentiation into PCs. IgE+ PCs exhibited dependency on the PI3K-mTOR axis that increased protein amounts of the transcription factor IRF4. In contrast, loss of components of the calcium-calcineurin-NFAT pathway promoted IgE+ PC differentiation. Mice bearing a B cell-specific deletion of calcineurin B1 exhibited increased production of IgE+ PCs. Mechanistically, sustained elevation of intracellular calcium in IgE+ PCs downstream of the IgE-BCR promoted BCL2L11-dependent apoptosis. Thus, chronic calcium signaling downstream of the IgE-BCR controls the self-limiting character of IgE responses and may be relevant to the accumulation of IgE-producing cells in allergic disease.

Calcineurin dephosphorylates topoisomerase IIß and regulates the formation of neuronal-activity-induced DNA breaks.

Neuronal activity induces topoisomerase IIß (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.

Recruitment of calcineurin to the TCR positively regulates T cell activation.

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (LckS59). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70Y493), as well as some downstream pathways in a manner consistent with signaling in cells expressing LckS59A (Lck that cannot be phosphorylated) or LckS59E (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

Sensing of ATP via the Purinergic Receptor P2RX7 Promotes CD8+ Trm Cell Generation by Enhancing Their Sensitivity to the Cytokine TGF-ß.

Tissue-resident memory (Trm) CD8+ T cells mediate protective immunity in barrier tissues, but the cues promoting Trm cell generation are poorly understood. Sensing of extracellular adenosine triphosphate (eATP) by the purinergic receptor P2RX7 is needed for recirculating CD8+ T cell memory, but its role for Trm cells is unclear. Here we showed that P2RX7 supported Trm cell generation by enhancing CD8+ T cell sensing of TGF-ß, which was necessary for tissue residency. P2RX7-deficient Trm cells progressively decayed in non-lymphoid tissues and expressed dysregulated Trm-specific markers. P2RX7 was required for efficient re-expression of the receptor TGF-ßRII through calcineurin signaling. Forced Tgfbr2 expression rescued P2RX7-deficient Trm cell generation, and TGF-ß sensitivity was dictated by P2RX7 agonists and antagonists. Forced Tgfbr2 also rescued P2RX7-deficient Trm cell mitochondrial function. Sustained P2RX7 signaling was required for long-term Trm cell maintenance, indicating that P2RX7 signaling drives induction and CD8+ T cell durability in barrier sites.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

Store-Operated Ca2+ Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.

Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.

Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

BACKGROUND: Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. METHODS: Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. RESULTS: We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. CONCLUSIONS: Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Nitro-oleic acid regulates T cell activation through post-translational modification of calcineurin.

Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inflammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modification of nucleophilic amino-acid residues. Several regulatory proteins have been identified as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inflammatory effects. Herein, we report the effects of nitro-oleic acid (NO2-OA) on pro-inflammatory T cell functions, showing that 9- and 10-NOA, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane, and IL-2, IL-4, and IFN-γ cytokine gene expressions. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates CaNA on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results provide evidence for an additional mechanism by which NO2-FAs exert their anti-inflammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses.

Octopamine metabolically reprograms astrocytes to confer neuroprotection against α-synuclein.

Octopamine is a well-established invertebrate neurotransmitter involved in fight or flight responses. In mammals, its function was replaced by epinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing in vitro and in vivo models of α-synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a trace amine-associated receptor 1-Orai1-Ca2+-calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the monocarboxylase transporter 2-calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by α-synuclein halt lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a unique function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to α-synucleinopathies.

Calcineurin and CK2 Reciprocally Regulate Synaptic AMPA Receptor Phenotypes via α2δ-1 in Spinal Excitatory Neurons.

Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506), are commonly used immunosuppressants for preserving transplanted organs and tissues. However, these drugs can cause severe and persistent pain. GluA2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) are implicated in various neurological disorders, including neuropathic pain. It is unclear whether and how constitutive calcineurin, a Ca2+/calmodulin protein phosphatase, controls synaptic CP-AMPARs. In this study, we found that blocking CP-AMPARs with IEM-1460 markedly reduced the amplitude of AMPAR-EPSCs in excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2), but not in inhibitory neurons expressing vesicular GABA transporter, in the spinal cord of FK506-treated male and female mice. FK506 treatment also caused an inward rectification in the current-voltage relationship of AMPAR-EPSCs specifically in VGluT2 neurons. Intrathecal injection of IEM-1460 rapidly alleviated pain hypersensitivity in FK506-treated mice. Furthermore, FK506 treatment substantially increased physical interaction of α2δ-1 with GluA1 and GluA2 in the spinal cord and reduced GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of spinal cords. Correspondingly, inhibiting α2δ-1 with pregabalin, Cacna2d1 genetic knock-out, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide reversed inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons caused by FK506 treatment. In addition, CK2 inhibition reversed FK506 treatment-induced pain hypersensitivity, α2δ-1 interactions with GluA1 and GluA2, and inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons. Thus, the increased prevalence of synaptic CP-AMPARs in spinal excitatory neurons plays a major role in calcineurin inhibitor-induced pain hypersensitivity. Calcineurin and CK2 antagonistically regulate postsynaptic CP-AMPARs through α2δ-1-mediated GluA1/GluA2 heteromeric assembly in the spinal dorsal horn.

Leishmania exploits host cAMP/EPAC/calcineurin signaling to induce an IL-33-mediated anti-inflammatory environment for the establishment of infection.

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis, and the pleiotropic cytokine interleukin (IL)-33 was found to be upregulated in infection. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated exchange protein activated by cAMP (EPAC)/calcineurin-dependent signaling and essential for the sustenance of infection. Leishmania donovani-infected macrophages showed upregulation of IL-33 and its neutralization resulted in decreased parasite survival and increased inflammatory responses. Infection-induced cAMP was involved in IL-33 production and of its downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 level. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin ensured nuclear localization of the transcription factors, nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha required for IL-33 transcription, and we further confirmed this by chromatin immunoprecipitation assay. Administering specific inhibitors of nuclear factor of activated T cell 1 and hypoxia-inducible factor 1 alpha in BALB/c mouse model of visceral leishmaniasis decreased liver and spleen parasite burden along with reduction in IL-33 level. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in tumor necrosis factor alpha and IL-12 level with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation.

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity; however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with the inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.

Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2 S production.

Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2 S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.

Calcineurin associates with centrosomes and regulates cilia length maintenance.

Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.

The immunosuppressive drug cyclosporin A has an immunostimulatory function in CD8+ T cells.

Cyclosporin A is a well-established immunosuppressive drug used to treat or prevent graft-versus-host disease, the rejection of organ transplants, autoimmune disorders, and leukemia. It exerts its immunosuppressive effects by inhibiting calcineurin-mediated dephosphorylation of the nuclear factor of activated T cells (NFAT), thus preventing its nuclear entry and suppressing T cell activation. Here we report an unexpected immunostimulatory effect of cyclosporin A in activating the mammalian target of rapamycin complex 1 (mTORC1), a crucial metabolic hub required for T cell activation. Through screening a panel of tool compounds known to regulate mTORC1 activation, we found that cyclosporin A activated mTORC1 in CD8+ T cells in a 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB/AKT)-dependent manner. Mechanistically, cyclosporin A inhibited the calcineurin-mediated AKT dephosphorylation, thereby stabilizing mTORC1 signaling. Cyclosporin A synergized with mTORC1 pathway inhibitors, leading to potent suppression of proliferation and cytokine production in CD8+ T cells and an increase in the killing of acute T cell leukemia cells. Consequently, relying solely on CsA is insufficient to achieve optimal therapeutic outcomes. It is necessary to simultaneously target both the calcineurin-NFAT pathway and the mTORC1 pathway to maximize therapeutic efficacy.