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FUS1/TUSC2 (FUSion1/TUmor Suppressor Candidate 2) is a tumor suppressor gene (TSG) originally described as a member of the TSG cluster from human 3p21.3 chromosomal region frequently deleted in lung cancer. Its role as a TSG in lung, breast, bone, and other cancers was demonstrated by several groups, but molecular mechanisms of its activities are starting to unveil lately. They suggest that Fus1-dependent mechanisms are relevant in etiologies of diseases beyond cancer, such as chronic inflammation, bacterial and viral infections, premature aging, and geriatric diseases. Here, we revisit the discovery of FUS1 gene in the context of tumor initiation and progression, and review 20 years of research into FUS1 functions and its molecular, structural, and biological aspects that have led to its use in clinical trials and gene therapy. We present a data-driven view on how interactions of Fus1 with the mitochondrial Ca2+ (mitoCa2+) transport machinery maintain cellular Ca2+ homeostasis and control cell apoptosis and senescence. This Fus1-mediated cellular homeostasis is at the crux of tumor suppressor, anti-inflammatory and anti-aging activities.

BackgroundCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and clinical evidence suggests that CDK4/6i can also promote antitumor T-cell responses. However, this pro-immunogenic property is yet to be successfully harnessed in the clinic, as combining CDK4/6i with immune checkpoint blockade (ICB) has not shown a definitive benefit in patients.MethodWe performed an in-depth analysis of the changes in the tumor immune microenvironment and systemic immune modulation associated with CDK4/6i treatment in muring breast cancer models and in patients with breast cancer using high dimensional flow cytometry and RNA sequencing. Gain and loss of function in vivo experiments employing cell transfer and depletion antibody were performed to uncover immune cell populations critical for CDK4/6i-mediated stimulation of antitumor immunity.ResultsWe found that loss of dendritic cells (DCs) within the tumor microenvironment resulting from CDK4/6 inhibition in bone marrow progenitors is a major factor limiting antitumor immunity after CDK4/6i and ICB. Consequently, restoration of DC compartment by adoptively transferring ex vivo differentiated DCs to mice treated with CDK4/6i and ICB therapy enabled robust tumor inhibition. Mechanistically, the addition of DCs promoted the induction of tumor-localized and systemic CD4 T-cell responses in mice receiving CDK4/6i-ICB-DC combination therapy, as characterized by enrichment of programmed cell death protein-1-negative T helper (Th)1 and Th2 cells with an activated phenotype. CD4 T-cell depletion abrogated the antitumor benefit of CDK4/6i-ICB-DC combination, with outgrowing tumors displaying an increased proportion of terminally exhausted CD8 T cells.ConclusionsOur findings suggest that CDK4/6i-mediated DC suppression limits CD4 T-cell responses essential for the sustained activity of CD8 T cells and tumor inhibition. Furthermore, they imply that restoring DC-CD4 T-cell crosstalk via DC transfer enables effective breast cancer immunity in response to CDK4/6i and ICB treatment.

FUS1/TUSC2 (FUSion1/TUmor Suppressor Candidate 2) is a tumor suppressor gene (TSG) originally described as a member of the TSG cluster from human 3p21.3 chromosomal region frequently deleted in lung cancer. Its role as a TSG in lung, breast, bone, and other cancers was demonstrated by several groups, but molecular mechanisms of its activities are starting to unveil lately. They suggest that Fus1-dependent mechanisms are relevant in etiologies of diseases beyond cancer, such as chronic inflammation, bacterial and viral infections, premature aging, and geriatric diseases. Here, we revisit the discovery of FUS1 gene in the context of tumor initiation and progression, and review 20 years of research into FUS1 functions and its molecular, structural, and biological aspects that have led to its use in clinical trials and gene therapy. We present a data-driven view on how interactions of Fus1 with the mitochondrial Ca

Inflammation is a major cause of morbidity and mortality in Western societies. Despite use of multiple drugs, both chronic and acute inflammation still represent major health burdens. Inflammation produces highly reactive dicarbonyl lipid peroxidation products such as isolevuglandins which covalently modify and cross-link proteins via lysine residues. Mitochondrial dysfunction has been associated with inflammation; however, its molecular mechanisms and pathophysiological role are still obscure. We hypothesized that inflammation-induced isolevuglandins contribute to mitochondrial dysfunction and mortality. To test this hypothesis, we have (a) investigated the mitochondrial dysfunction in response to synthetic 15-E2-isolevuglandin (IsoLG) and its adducts; (b) developed a new mitochondria-targeted scavenger of isolevuglandins by conjugating 2-hydroxybenzylamine to the lipophilic cation triphenylphosphonium, (4-(4-aminomethyl)-3-hydroxyphenoxy)butyl)-triphenylphosphonium (mito2HOBA); (c) tested if mito2HOBA protects from mitochondrial dysfunction and mortality using a lipopolysaccharide model of inflammation. Acute exposure to either IsoLG or IsoLG adducts with lysine, ethanolamine or phosphatidylethanolamine inhibits mitochondrial respiration and attenuates Complex I activity. Complex II function was much more resistant to IsoLG. We confirmed that mito2HOBA markedly accumulates in isolated mitochondria and it is highly reactive with IsoLGs. To test the role of mitochondrial IsoLGs, we studied the therapeutic potential of mito2HOBA in lipopolysaccharide mouse model of sepsis. Mito2HOBA supplementation in drinking water (0.1 g/L) to lipopolysaccharide treated mice increased survival by 3-fold, improved complex I-mediated respiration, and histopathological analyses supported mito2HOBA-mediated protection of renal cortex from cell injury. These data support the role of mitochondrial IsoLG in mitochondrial dysfunction and inflammation. We conclude that reducing mitochondrial IsoLGs may be a promising therapeutic target in inflammation and conditions associated with mitochondrial oxidative stress and dysfunction., Graphical abstract Image 1, Highlights • Isolevuglandins and their adducts inhibit mitochondrial respiration and attenuates Complex I activity. • 2-Hydroxybenzylamine conjugated to triphenylphosphonium, mito2HOBA, accumulates in mitochondria. • Mito2HOBA in drinking water improves complex I-mediated respiration in LPS model of sepsis. • Mitochondria-targeted scavenger of isolevuglandins mito2HOBA reduces mortality in LPS model.

Many melanomas are resistant to standard-of-care immune checkpoint blockade and inhibitors of mutated BRAF. New effective therapies that work via immune and BRAF-independent mechanisms are urgently needed. We have previously demonstrated that an inhibitor of mitotic kinase Aurora A (AURKAi) can induce a distinct type of stable cell cycle known as senescence in melanoma tumors independent of their genetic background. Here we hypothesized that combination of AURKAi with a drug that selectively kills senescent cells, such as BCL2 inhibitor (BCLi) navitoclax, will be synthetically lethal to melanoma cells. Melanoma patient-derived organoids (PDOs) were used to test efficacy of AURKAi and BCLi combination treatment. To generate organoids, patient-derived tumor cells were cultured ex vivo in 3D matrix. Drug response was evaluated using fluorescent viability assay. Fifteen out of twenty PDOs were highly sensitive to AURKAi and BCLi combination treatment, exhibiting robust induction of cell death. No cell death was observed when either drug was added individually. NextGen DNA sequencing indicated that all sensitive PDOs retained wild-type TP53, while resistant tumors had mutations in this gene. Knockout or knockdown of TP53 abrogated AURKAi and BCLi-induced cell death in melanoma cells, suggesting that transcription factor p53 encoded by TP53 is a key mediator of drug response. Mechanistically, p53 was induced by AURKAi treatment independent of BCLi. However, single-agent AURKAi treatment resulted in activation of p53-mediated cell cycle arrest based on increased expression of CDK inhibitor p21 and decreased expression of proliferation markers. In contrast, addition of BCLi to AURKAi activated proapoptotic p53 target BAX and induced cleavage of PARP and caspases 3, 7, and 8. Treatment with pan-caspase inhibitor abrogated cell death, suggesting caspase-dependent apoptosis. Electron microscopy revealed morphologic signs of apoptosis in combination-treated cells, including cell blebbing, apoptotic bodies, and nuclear fragmentation. This suggests that BCLi shifts cell fate decision in AURKAi-treated cells towards apoptosis. In conclusion, combination treatment with AURKAi and BCl2i induced p53-dependent apoptosis. Preclinical evaluation of this drug combination in PDO model demonstrated robust efficacy against melanomas with wild-type p53. Since p53 mutations are relatively rare in melanoma, a combination of AURKAi and BCLi presents a promising therapeutic option for this deadly disease. Citation Format: Vijaya Bharti, Ashlyn Blevins, Vivian L. Weiss, Sheau C Chen, Roman Uzhachenko, Ann Richmond, Anna E. Vilgelm. Patient-derived organoids demonstrate synergistic effect of co-targeting Aurora kinase and prosurvival BCL2 family proteins [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B20.

The interaction between the nervous system and immune system has sparked interest in recent years. Emerging evidence shows an intricate neuroimmune network. We were intrigued by the expression of various neurotransmitter receptors on T lymphocytes. Specifically, following TCR stimulation, glutamate receptors (GluR) were significantly upregulated on both CD4+ and CD8+ T cells, with a peak at 48 h. Concomitant with the upregulation of activation molecules CD69, CD25 and CD44, proliferating CD8+ T cells presented higher levels of GluA3 and mGluR1 when compared with resting cells. By blocking group I metabotropic glutamate receptors and AMPA receptors through antagonists, we show that CD8+ T cells have a delayed activation but their viability is not altered. However, GluR blockade affected CD8+ T cell proliferation and their ability to kill tumor cells in vivo or target cells in vitro. While the frequency of GluR+CD8+ T cells ranges around 30% of total CD8+ T population, the effect of blocking glutamate signaling through receptor antagonists is a dominant effect and it is mediated by transient reduction in protein phosphorylation of TCR mediated pathways as well as calcium modulation. Overall, data suggest that glutamate receptors may have a stimulatory effect on T cell activation and glutamate agonists may boost T cell response in an immunosuppressive setting such as cancer.

Rationale: Clinical studies have shown that Sirt3 (Sirtuin 3) expression declines by 40% by 65 years of age paralleling the increased incidence of hypertension and metabolic conditions further inactivate Sirt3 because of increased NADH (nicotinamide adenine dinucleotide, reduced form) and acetyl-CoA levels. Sirt3 impairment reduces the activity of a key mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2) because of hyperacetylation. Objective: In this study, we examined whether the loss of Sirt3 activity increases vascular oxidative stress because of SOD2 hyperacetylation and promotes endothelial dysfunction and hypertension. Methods and Results: Hypertension was markedly increased in Sirt3-knockout (Sirt3 −/ − ) and SOD2-depleted (SOD2 +/ − ) mice in response to low dose of angiotensin II (0.3 mg/kg per day) compared with wild-type C57Bl/6J mice. Sirt3 depletion increased SOD2 acetylation, elevated mitochondrial O 2 · – , and diminished endothelial nitric oxide. Angiotensin II-induced hypertension was associated with Sirt3 S-glutathionylation, acetylation of vascular SOD2, and reduced SOD2 activity. Scavenging of mitochondrial H 2 O 2 in mCAT mice expressing mitochondria-targeted catalase prevented Sirt3 and SOD2 impairment and attenuated hypertension. Treatment of mice after onset of hypertension with a mitochondria-targeted H 2 O 2 scavenger, mitochondria-targeted hydrogen peroxide scavenger ebselen, reduced Sirt3 S-glutathionylation, diminished SOD2 acetylation, and reduced blood pressure in wild-type but not in Sirt3 −/− mice, whereas an SOD2 mimetic, (2-[2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino]-2-oxoethyl) triphenylphosphonium (mitoTEMPO), reduced blood pressure and improved vasorelaxation both in Sirt3 −/− and wild-type mice. SOD2 acetylation had an inverse correlation with SOD2 activity and a direct correlation with the severity of hypertension. Analysis of human subjects with essential hypertension showed 2.6-fold increase in SOD2 acetylation and 1.4-fold decrease in Sirt3 levels, whereas SOD2 expression was not affected. Conclusions: Our data suggest that diminished Sirt3 expression and redox inactivation of Sirt3 lead to SOD2 inactivation and contributes to the pathogenesis of hypertension.

Endothelial dysfunction is associated with aging, diabetes, hyperlipidemia, obesity and these risk factors affect the expression and activity of the mitochondrial deacetylase Sirt3. Sirt3 activates major antioxidant SOD2 by deacetylation of specific lysine residues and Sirt3 depletion increases oxidative stress. We hypothesized that loss of vascular Sirt3 increases endothelial dysfunction, promotes hypertension and end organ damage. The role of vascular Sirt3 was studied in wild-type C57Bl/6J mice and tamoxifen-inducible smooth muscle specific Sirt3 knockout mice (Smc Sirt3 KO ) using angiotensin II model of hypertension (Ang II, 0.7 mg/kg/day). Western blot showed 30% reduction of vascular Sirt3 and 2-fold increase in SOD2 acetylation in Ang II-infused WT mice. We have tested if ex vivo treatment of aorta with Sirt3 activator resveratrol improves endothelial function. Indeed, ex vivo incubation with resveratrol (10 μM) significantly reduced SOD2 acetylation, diminished mitochondrial O 2 and increased endothelial NO to normal level while Sirt3-inactive analog dihydroresveratrol had no effect. Specific role of vascular Sirt3 was studied in Smc Sirt3 KO mice by crossing floxed Sirt3 mice with mice carrying gene for inducible cre in the vascular smooth muscle. Sirt3 deletion exacerbates hypertension (165 mm Hg vs 155 mm Hg in wild-type) and significantly increases mortality in Ang II-infused Smc Sirt3 KO mice (60% vs 10% in wild-type) associated with severe edema and aortic aneurysm (100% vs 20% in wild-type). Decrease of NO is a hallmark of endothelial dysfunction in hypertension due to vascular oxidative stress. Indeed, Ang II infusion increased vascular O 2 by 2-fold and reduced endothelial NO by 2-fold. Interestingly, Ang II infusion in Smc Sirt3 KO mice caused severe vascular oxidative stress (3-fold increase in O 2 ) and exacerbated endothelial dysfunction (4-fold decrease in NO). These data indicate that reduced vascular Sirt3 activity occurs in hypertension and this promotes vascular oxidative stress, increases endothelial dysfunction, exacerbates hypertension, increases end-organ-damage and mortality. It is conceivable that Sirt3 agonists and SOD2 mimetics may have therapeutic potential in cardiovascular disease.

Fus1, encoded by a 3p21.3 tumour suppressor gene, is down-regulated, mutated or lost in the majority of inflammatory thoracic malignancies. The mitochondrial localization of Fus1 stimulated us to investigate how Fus1 modulates inflammatory response and mitochondrial function in a mouse model of asbestos-induced peritoneal inflammation. Asbestos treatment resulted in a decreased Fus1 expression in wild-type (WT) peritoneal immune cells, suggesting that asbestos exposure may compromise the Fus1-mediated inflammatory response. Untreated Fus1(-/-) mice had an ~eight-fold higher proportion of peritoneal granulocytes than Fus1(+/+) mice, pointing at ongoing chronic inflammation. Fus1(-/-) mice exhibited a perturbed inflammatory response to asbestos, reflected in decreased immune organ weight and peritoneal fluid protein concentration, along with an increased proportion of peritoneal macrophages. Fus1(-/-) immune cells showed augmented asbestos-induced activation of key inflammatory, anti-oxidant and genotoxic stress response proteins ERK1/2, NFκB, SOD2, γH2AX, etc. Moreover, Fus1(-/-) mice demonstrated altered dynamics of pro- and anti-inflammatory cytokine expression, such as IFNγ, TNFα, IL-1A, IL-1B and IL-10. 'Late' response cytokine Ccl5 was persistently under-expressed in Fus1(-/-) immune cells at both basal and asbestos-activated states. We observed an asbestos-related difference in the size of CD3(+) CD4(-) CD8(-) DN T cell subset that was expanded four-fold in Fus1(-/-) mice. Finally, we demonstrated Fus1-dependent basal and asbestos-induced changes in major mitochondrial parameters (ROS production, mitochondrial potential and UCP2 expression) in Fus1(-/-) immune cells and in Fus1-depleted cancer cells, thus supporting our hypothesis that Fus1 establishes its immune- and tumour-suppressive activities via regulation of mitochondrial homeostasis.

A large majority of immune-oncology research efforts focus on T-cell immunotherapy. However, limited clinical efficacy has been achieved with adoptive T-cell effectors alone. In addition, research has shown that the sole use of adoptive T-cells leads to the development of tumor escape variants and “occult cancer”. Conversely, tumor-infiltrating natural killer (NK) cells have been shown to cause elimination of these escape variants. These observations indicate that the interaction between the innate and adaptive immune effectors is indispensable for effective tumor immunity. Notch is a major signaling system important for communication between neighboring cells. Our previous studies show that it plays a crucial role in the differentiation and antitumor function of CD8+T cells. However, its role in NK cell activation and antitumor function is not clear. Thus, we analyzed Notch receptor-ligand system on NK cells and in the context of their crosstalk with CD8+ T cells. Our data indicate that inhibiting Notch signaling in NK cells decreases NK cell activation. In addition, Notch intracellular domain (NICD) expression is increased upon NK activation, suggesting that the Notch signaling pathway may be involved in NK activation. Our data also show that activated CD8+T cells activate naïve NK cells through direct physical contact. Transmembrane notch receptor expression is decreased during this interaction suggesting that the Notch receptors may be involved in CD8+T cell – NK cell crosstalk. Experiments are underway to further understand the relevance of Notch signaling pathway in innate-adaptive immune crosstalk. The findings will help develop novel strategies for adoptive cell immunotherapies, specifically against solid tumors.

The interaction between the nervous system and immune system has sparked interest in recent years. Emerging evidence shows an intricate neuro-immune network. Studies revealed that neurotransmitters can trigger immune cell responses such as cytokine secretion, integrin expression, and chemotaxis. We were intrigued by the expression of various neurotransmitter receptors on mouse T lymphocytes. Specifically, following TCR stimulation, glutamate receptors (GluR) were significantly upregulated on both CD4+ and CD8+T cells, with a peak at 48 h. Concomitant with the upregulation of activation molecules CD69, CD25 and CD44, proliferating CD8+T cells presented higher levels of GluR3 and GluR1 when compared with non-proliferating cells, as well as a higher production of IFN-g and granzyme B. Based on the augmentation of GluR expression upon T cell activation, we evaluated the role of GluR signaling in modulating T cells in orthotopic mammary tumors that express a low avidity epitope of a model antigen hemagglutinin (4T1HA). We found that HA-reactive CD8+ T cells in the tumor-infiltrate had an upregulated expression of GluR3 and GluR1 when compared with control group. The blockade of GluR signaling down-regulated expression of CD69 and CD25, impaired T cell proliferation, and interfered with their cytolytic activity. Early TCR-activation pathways such as Lck/Akt as well as calcium signaling were affected by GluR antagonists, suggesting their signaling interaction. Overall, data suggest that glutamate receptors may have costimulatory effects on T cell activation and glutamate agonists may boost T cell response in an immunosuppressive setting such as cancer.

Unraveling the complexity of lymphocyte interactions is key to understanding immune network and improving cancer immunotherapy. By designing various adoptive cell transfer protocols in solid tumors established in RAG−/−, RAG−/−gc−/− and RAG−/−GzmB-Tom mice and tracing lymphocytes specific to a self-tumor antigen P1A encoded by an X-linked cancer-germline gene Trap1a, we explored the functional dynamics between T and NK cells. We found that activated CD8+T cells augmented NK cell effector function in a pre-tumor T cell protocol that prevented the development of antigen-escape tumor variants. Using a silica nanofiber 3D matrix to control cellular mobility, confocal imaging showed intercellular contacts of activated CD8+T cells (CD69highCD25high) with multiple naïve NK cells, while naïve CD8+T cells formed single or no contact with NK cells. This interaction led to crossregulation of mitochondrial Ca2+ (mitoCa2+) oscillations in both cells whereby activated CD8+T cells induced differentiation of NK cell effector phenotype and NK cells polarized CD8+T cells towards a central memory phenotype. Intracellularly, CD8+T cells increased JAK1, JAK3, TYK2, STAT2 and STAT6 phosphorylation and oxidative signaling in NK cells. In turn, NK cells restrained IL-2 signaling in CD8+T cells by dampening activation-induced STAT5-dependent expression of IL-2-receptor-a chain. These effects were abrogated following the blockade of mitoCa2+ uptake. Mice deficient in mitoCa2+ handling-regulatory gene Fus1 showed increased incidence of a range of spontaneous sarcomas, lymphomas and leukemia. Data suggest that mitoCa2+ transport-guided intercellular crosstalk between CD8+T and NK cells is critical to prevent tumor development and escape.

// Roman Uzhachenko 1 , Anil Shanker 1, 2 , Wendell G. Yarbrough 3, 4, 5 , Alla V. Ivanova 3 1 Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, Tennessee, United States 2 Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University, Nashville, Tennessee, United States 3 Department of Surgery, Division of Otolaryngology, Yale School of Medicine, New Haven, Connecticut, United States 4 Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, United States 5 Department of Pathology, Yale School of Medicine, New Haven, Connecticut, United States Correspondence to: Alla V. Ivanova, e-mail: alla.ivanova@yale.edu Keywords: Fus1/Tusc2, mitochondria, calcium, tumor suppressor, inflammation, autoimmunity Received: March 23, 2015 Accepted: June 27, 2015 Published: July 15, 2015 ABSTRACT Mitochondria present a unique set of key intracellular functions such as ATP synthesis, production of reactive oxygen species (ROS) and Ca 2+ buffering. Mitochondria both encode and decode Ca 2+ signals and these interrelated functions have a direct impact on cell signaling and metabolism. High proliferative potential is a key energy-demanding feature shared by cancer cells and activated T lymphocytes. Switch of a metabolic state mediated by alterations in mitochondrial homeostasis plays a fundamental role in maintenance of the proliferative state. Recent studies show that tumor suppressors have the ability to affect mitochondrial homeostasis controlling both cancer and autoimmunity. Herein, we discuss established and putative mechanisms of calcium–dependent regulation of both T cell and tumor cell activities. We use the mitochondrial protein Fus1 as a case of tumor suppressor that controls immune response and tumor growth via maintenance of mitochondrial homeostasis. We focus on the regulation of mitochondrial Ca 2+ handling as a key function of Fus1 and highlight the mechanisms of a crosstalk between Ca 2+ accumulation and mitochondrial homeostasis. Given the important role of Ca 2+ signaling, mitochondrial Ca 2+ transport and ROS production in the activation of NFAT and NF-κB transcription factors, we outline the importance of Fus1 activities in this context.

Endothelial dysfunction is associated with aging, diabetes, hyperlipidemia, obesity and these risk factors affect the expression and activity of the mitochondrial energy regulator deacetylase Sirt3. Sirt3 activates major mitochondrial antioxidant enzyme SOD2 by deacetylation of specific lysine residues and Sirt3 depletion increases oxidative stress. We hypothesized that loss of vascular Sirt3 activity increases vascular oxidative stress and endothelial dysfunction and that this promotes hypertension and end organ damage. The role of vascular Sirt3 was studied in wild-type C57Bl/6J mice and tamoxifen-inducible smooth muscle specific Sirt3 knockout mice (Smc Sirt3KO ) using angiotensin II model of hypertension (Ang II, 0.7 mg/kg/day). Western blot analysis of aorta showed 30% reduction of vascular Sirt3 and increased SOD2 acetylation by 2-fold after onset of hypertension. We have tested if ex vivo treatment of aorta with Sirt3 activator resveratrol reduces SOD2 acetylation, diminishes mitochondrial O 2 ●– , and increases the production of endothelial NO. Indeed, however, ex vivo incubation with resveratrol (10 µM) significantly reduced SOD2 acetylation, diminished mitochondrial O 2 ●– and increased endothelial NO to normal level while Sirt3-inactive analog dihydroresveratrol had no effect. Specific role of vascular Sirt3 was studied in Smc Sirt3KO mice by crossing floxed Sirt3 mice with mice carrying gene for inducible cre in the vascular smooth muscle (inducible SMMHC cre). Wild-type and Smc Sirt3KO mice were injected daily for 5 days with tamoxifen and Ang II osmotic pumps were installed 10 days later. Deletion of Sirt3 in smooth muscle exacerbates hypertension (165 mm Hg vs 155 mm Hg in wild-type) and significantly increased mortality in Ang II-infused Smc Sirt3KO mice (60% vs 10% in wild-type) associated with severe edema and aortic aneurysm formation (100% vs 20% in wild-type). Decrease of NO is a hallmark of endothelial dysfunction in hypertension due to vascular oxidative stress. Indeed, Ang II infusion increased vascular O 2 ●– by 2-fold and reduced endothelial NO by 2-fold. Interestingly, Ang II infusion in Smc Sirt3KO mice caused severe vascular oxidative stress (3-fold increase in O 2 ●– ) and exacerbated endothelial dysfunction (4-fold decrease in NO). These data indicate that reduced vascular Sirt3 activity occurs in hypertension and this leads to SOD2 hyperacetylation and inactivation, promotes vascular oxidative stress, increases endothelial dysfunction, exacerbates hypertension, increases end-organ-damage and mortality. It is conceivable that Sirt3 agonists and SOD2 mimetics may have therapeutic potential in cardiovascular disease.

Free radical oxidation of arachidonic acid produces highly reactive isolevuglandins (isoLG) which causes mitochondrial dysfunction by opening of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP regulatory subunit cyclophilin D reduces isoLG-induced mitochondrial dysfunction (Free Radic Biol Med 2010;49:567-79). We found that depletion of cyclophilin D diminishes mitochondrial O 2 ●– , improves vascular relaxation and reduces hypertension (Hypertension 2016;67:1218-27). We hypothesized that hypertension is associated with accumulation of mitochondrial isoLG and mitochondria-targeted isoLG scavenger reduces vascular oxidative stress and attenuates hypertension. To test this hypothesis we developed mitochondria-targeted isoLG scavenger 4-(aminomethyl)-3-hydroxyphenoxy) butyl)triphenyl-phosphonium (mito2HOBA) by conjugation of lipophilic triphenylmethylphosphonium cation to 2-hydroxybenzylamine (2HOBA). Mito2HOBA is a water soluble compound which was well tolerated by cultured human aortic endothelial cells (HAECs) and by mice receiving it in drinking water. It was found that mito2HOBA (50 nM) inhibited mitochondrial O 2 ●– production (MitoSOX/HPLC) and prevented cardiolipin oxidation (LS-MS) in HEACs incubated with TNFα+Angiotensin II while untargeted analog 2HOBA (50 nM) was not effective. The functional role of mitochondrial isoLG was tested in vivo using angiotensin II model of hypertension. C57Bl/6J mice were infused with angiotensin II (0.7 mg/kg/day) or saline (Sham) and received mito2HOBA in the drinking water (0.1 g/L). It was found that mito2HOBA diminished angiotensin II-induced hypertension. Hypertension was associated with increased formation of mitochondrial isoLG measured by Western blot analysis of heart mitochondria using the anti-isoLG D11 scfv antibody and 2-fold decrease in kidney ATP. mito2HOBA reduced accumulation of isoLG adducts in the heart mitochondria and prevented drop in kidney ATP in angiotensin II-infused mice. Decrease of NO is a hallmark of endothelial dysfunction in hypertension due to vascular oxidative stress. We examined vascular oxidative stress by measurements of aortic O 2 ●– using fluorescent O 2 ●– probe DHE and HPLC, and analysis of endothelial NO using Electron Spin Resonance and specific NO spin trap Fe(DETC) 2 . It was found that mito2HOBA reduced vascular O 2 ●– in angiotensin II-infused mice and preserved endothelial NO. These data support the role of mitochondrial oxidative stress and mitochondrial isoLG in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoLG may have therapeutic potential in cardiovascular disease.

Fus1, encoded by a 3p21.3 tumour suppressor gene, is down-regulated, mutated or lost in the majority of inflammatory thoracic malignancies. The mitochondrial localization of Fus1 stimulated us to investigate how Fus1 modulates inflammatory response and mitochondrial function in a mouse model of asbestos-induced peritoneal inflammation. Asbestos treatment resulted in a decreased Fus1 expression in wild-type (WT) peritoneal immune cells, suggesting that asbestos exposure may compromise the Fus1-mediated inflammatory response. Untreated Fus1(-/-) mice had an ~eight-fold higher proportion of peritoneal granulocytes than Fus1(+/+) mice, pointing at ongoing chronic inflammation. Fus1(-/-) mice exhibited a perturbed inflammatory response to asbestos, reflected in decreased immune organ weight and peritoneal fluid protein concentration, along with an increased proportion of peritoneal macrophages. Fus1(-/-) immune cells showed augmented asbestos-induced activation of key inflammatory, anti-oxidant and genotoxic stress response proteins ERK1/2, NFκB, SOD2, γH2AX, etc. Moreover, Fus1(-/-) mice demonstrated altered dynamics of pro- and anti-inflammatory cytokine expression, such as IFNγ, TNFα, IL-1A, IL-1B and IL-10. 'Late' response cytokine Ccl5 was persistently under-expressed in Fus1(-/-) immune cells at both basal and asbestos-activated states. We observed an asbestos-related difference in the size of CD3(+) CD4(-) CD8(-) DN T cell subset that was expanded four-fold in Fus1(-/-) mice. Finally, we demonstrated Fus1-dependent basal and asbestos-induced changes in major mitochondrial parameters (ROS production, mitochondrial potential and UCP2 expression) in Fus1(-/-) immune cells and in Fus1-depleted cancer cells, thus supporting our hypothesis that Fus1 establishes its immune- and tumour-suppressive activities via regulation of mitochondrial homeostasis.

Local tumor antigen-specific T cell-NK cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of the T cell responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+ T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-γc-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. In 2D and 3D co-culture systems, we observed that PMA/ionomycin-stimulated CD8+ T cells form multiple contacts with naïve NK lymphocytes. Data show that NK cells interacting with activated CD8+ T cells show an up-regulation of CD25 and CD69 expression mediated by intercellular contacts, and activation of NKG2D receptors and Stat2, Stat6, Jak1, Jak3, Tyk2, and PTEN signaling molecules with a decrease in the phosphorylation of Stat1, PKB/Akt, SAPK/JNK, p38. On the other hand, interacting NK cells down-regulate CD25 molecule expression on CD8+ T cells and promote differentiation of central memory CD44+CD62L+ T cells. CD8+ T cells display an elevation in the phosphorylation of Stat1 and down-regulation of Stat5 with stimulated PKB/Akt, Lck, mTOR, and p42/p44. Moreover, significant changes in the cytosolic and mitochondrial Ca2+, production of mitochondrial ROS, mitochondrial membrane potential, mitochondrial permeability transition pore, and synthesis of nitric oxide and non-protein thiols (mostly, reduced glutathion) were observed in a reciprocal T cell-NK cell interaction. These results highlight the importance of mitochondrial activity in the re-modeling of activation signaling and memory differentiation of interacting CD8 T cells and NK cells. These results will help refine cancer immunotherapeutic strategies. Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Crosstalk between CD8+ T and NK cells: fine-tuning of antitumor immune response. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2015-4059