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Background Translocation of miR‐181c into cardiac mitochondria downregulates the mitochondrial gene, mt‐ COX 1. miR‐181c/d −/− hearts experience less oxidative stress during ischemia/reperfusion (I/R) and are protected against I/R injury. Additionally, miR‐181c overexpression can increase mitochondrial matrix Ca 2+ ([Ca 2+ ] m ), but the mechanism by which miR‐181c regulates [Ca 2+ ] m is unknown. Methods and Results By RNA sequencing and analysis, here we show that hearts from miR‐181c/d −/− mice overexpress nuclear‐encoded Ca 2+ regulatory and metabolic pathway genes, suggesting that alterations in miR‐181c and mt‐ COX 1 perturb mitochondria‐to‐nucleus retrograde signaling and [Ca 2+ ] m regulation. Quantitative polymerase chain reaction validation of transcription factors that are known to initiate retrograde signaling revealed significantly higher Sp1 (specificity protein) expression in the miR‐181c/d −/− hearts. Furthermore, an association of Sp1 with the promoter region of MICU 1 was confirmed by chromatin immunoprecipitation‐quantitative polymerase chain reaction and higher expression of MICU 1 was found in the miR‐181c/d −/− hearts. Conversely, downregulation of Sp1 by small interfering RNA decreased MICU 1 expression in neonatal mouse ventricular myocytes. Changes in PDH activity provided evidence for a change in [Ca 2+ ] m via the miR‐181c/ MICU 1 axis. Moreover, this mechanism was implicated in the pathology of I/R injury. When MICU 1 was knocked down in the miR‐181c/d −/− heart by lentiviral expression of a short‐hairpin RNA against MICU 1, cardioprotective effects against I/R injury were abrogated. Furthermore, using an in vitro I/R model in miR‐181c/d −/− neonatal mouse ventricular myocytes, we confirmed the contribution of both Sp1 and MICU 1 in ischemic injury. Conclusions miR‐181c regulates mt‐ COX 1, which in turn regulates MICU 1 expression through the Sp1‐mediated mitochondria‐to‐nucleus retrograde pathway. Loss of miR‐181c can protect the heart from I/R injury by modulating [Ca 2+ ] m through the upregulation of MICU 1.

Arsenic is a common contaminant in drinking water throughout the world, and recent studies support a link between inorganic arsenic (iAS) exposure and ischemic heart disease in men and women. Female hearts exhibit an estrogen-dependent reduction in susceptibility to myocardial ischemic injury compared with males, and as such, female hearts may be more susceptible to the endocrine-disrupting effects of iAS exposure. However, iAS exposure and susceptibility to ischemic heart injury have not been examined in mechanistic studies. Male and female mice (8 wk) were exposed to environmentally relevant concentrations of sodium arsenite (0, 10, 100, and 1,000 parts/billion) via drinking water for 4 wk. Pre- and postexposure echocardiography was performed, and postexposure plasma was collected for 17β-estradiol measurement. Hearts were excised and subjected to ischemia-reperfusion (I/R) injury via Langendorff perfusion. Exposure to 1,000 parts/billion iAS led to sex-disparate effects, such that I/R injury was exacerbated in female hearts but unexpectedly attenuated in males. Assessment of echocardiographic parameters revealed statistically significant structural remodeling in iAS-treated female hearts with no change in function; males showed no change. Plasma 17β-estradiol levels were not significantly altered by iAS in male or female mice versus nontreated controls. Although total eNOS protein levels did not change in whole heart homogenates from iAS-treated male or female mice, eNOS phosphorylation (Ser1177) was significantly elevated in iAS-treated male hearts. These results suggest that iAS exposure can induce sex-disparate effects and modulate susceptibility to ischemic heart injury by targeting distinct sex-dependent pathways. NEW & NOTEWORTHY This is the first mechanistic study examining iAS exposure on myocardial ischemia-reperfusion injury in male and female mice. Following iAS exposure, ischemia-reperfusion injury was exacerbated in female hearts but attenuated in males. iAS treatment induced statistically significant cardiac remodeling in females, with no change in males. iAS treatment also enhanced phosphorylated eNOS levels at Ser1177, but only in male hearts. These results suggest that iAS alters susceptibility to myocardial I/R injury through distinct sex-dependent pathways.

Rationale: Protein S -nitros(yl)ation (SNO) has been implicated as an essential mediator of nitric oxide-dependent cardioprotection. Compared with males, female hearts exhibit higher baseline levels of protein SNO and associated with this, reduced susceptibility to myocardial ischemia-reperfusion injury. Female hearts also exhibit enhanced S -nitrosoglutathione reductase (GSNO-R) activity, which would typically favor decreased SNO levels as GSNO-R mediates SNO catabolism. Objective: Because female hearts exhibit higher SNO levels, we hypothesized that GSNO-R is an essential component of sex-dependent cardioprotection in females. Methods and Results: Male and female wild-type mouse hearts were subjected to ex vivo ischemia-reperfusion injury with or without GSNO-R inhibition (N6022). Control female hearts exhibited enhanced functional recovery and decreased infarct size versus control males. Interestingly, GSNO-R inhibition reversed this sex disparity, significantly reducing injury in male hearts, and exacerbating injury in females. Similar results were obtained with male and female GSNO-R −/− hearts using ex vivo and in vivo models of ischemia-reperfusion injury. Assessment of SNO levels using SNO-resin assisted capture revealed an increase in total SNO levels with GSNO-R inhibition in males, whereas total SNO levels remained unchanged in females. However, we found that although GSNO-R inhibition significantly increased SNO at the cardioprotective Cys39 residue of nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 in males, SNO-NADH dehydrogenase subunit 3 levels were surprisingly reduced in N6022-treated female hearts. Because GSNO-R also acts as a formaldehyde dehydrogenase, we examined postischemic formaldehyde levels and found that they were nearly 2-fold higher in N6022-treated female hearts compared with nontreated hearts. Importantly, the mitochondrial aldehyde dehydrogenase 2 activator, Alda-1, rescued the phenotype in GSNO-R −/− female hearts, significantly reducing infarct size. Conclusions: These striking findings point to GSNO-R as a critical sex-dependent mediator of myocardial protein SNO and formaldehyde levels and further suggest that different therapeutic strategies may be required to combat ischemic heart disease in males and females.

Acute myeloid leukemia (AML) is a devastating disease with poor patient survival. As targetable mutations in AML are rare, novel oncogenic mechanisms are needed to define new therapeutic targets. We identified AML cells that exhibit an aberrant pool of nuclear glycogen synthase kinase 3β (GSK3β). This nuclear fraction drives AML growth and drug resistance. Nuclear, but not cytoplasmic, GSK3β enhances AML colony formation and AML growth in mouse models. Nuclear GSK3β drives AML partially by promoting nuclear localization of the NF-κB subunit, p65. Finally, nuclear GSK3β localization has clinical significance as it strongly correlates to worse patient survival (n = 86; hazard ratio = 2.2; P < .01) and mediates drug resistance in cell and animal models. Nuclear localization of GSK3β may define a novel oncogenic mechanism in AML and represent a new therapeutic target.

Natural killer cells harnessed from healthy individuals can be expanded ex vivo using various platforms to produce large doses for adoptive transfer into cancer patients. During such expansion, NK cells are increasingly activated and more efficient at killing cancer cells. Adoptive transfer however introduces these activated cells into a highly immunosuppressive tumor microenvironment mediated in part by excessive transforming growth factor beta (TGF-beta) from both cancer cells and their surrounding stroma. This microenvironment ultimately limits the clinical efficacy of NK cell therapy. In this study, we examined the use of a TGF-beta receptor kinase inhibitor, LY2157299, in preserving the cytotoxic function of ex vivo expanded, highly activated NK cells following sustained exposure to pathologic levels of TGF-beta in vitro and in a liver metastases model of colon cancer. Using myeloid leukemia and colon cancer cell lines, we show that the TGF-beta driven impairment of NK cell cytotoxicity is mitigated by LY2157299. We demonstrate this effect using quantitative cytotoxicity assays as well as by showing a preserved activated phenotype with high NKG2D/CD16 expression and enhanced cytokine production. In a mouse liver metastases model of colon cancer, we observed significantly improved eradication of liver metastases in mice treated with adoptive NK cells combined with LY2157299 compared with mice receiving NK cells or TGF beta inhibition alone. We propose that the therapeutic efficacy of adoptive NK cell therapy clinically will be markedly enhanced by complementary approaches targeting TGF-beta signaling in vivo.

Nitric oxide (NO) plays an important role in cardioprotection, and recent work from our group and others has implicated protein S-nitrosylation (SNO) as a critical component of NO-mediated protection in different models, including ischemic pre- and post-conditioning and sex-dependent cardioprotection. However, studies have yet to examine whether protein SNO levels are similarly increased with pharmacologic preconditioning in male and female hearts, and whether an increase in protein SNO levels, which is protective in male hearts, is sufficient to increase baseline protection in female hearts. Therefore, we pharmacologically preconditioned male and female hearts with the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA). CHA administration prior to ischemia significantly improved functional recovery in both male and female hearts compared to baseline in a Langendorff-perfused heart model of ischemia-reperfusion injury (% of preischemic function ± SE: male baseline: 37.5±3.4% vs. male CHA: 55.3±3.2%; female baseline: 61.4±5.7% vs. female CHA: 76.0±6.2%). In a separate set of hearts, we found that CHA increased p-Akt and p-eNOS levels. We also used SNO-resin-assisted capture with LC-MS/MS to identify SNO proteins in male and female hearts, and determined that CHA perfusion induced a modest increase in protein SNO levels in both male (11.4%) and female (12.3%) hearts compared to baseline. These findings support a potential role for protein SNO in a model of pharmacologic preconditioning, and provide evidence to suggest that a modest increase in protein SNO levels is sufficient to protect both male and female hearts from ischemic injury. In addition, a number of the SNO proteins identified with CHA treatment were also observed with other forms of cardioprotective stimuli in prior studies, further supporting a role for protein SNO in cardioprotection.

Introduction: The tumor micro-environment poses a limitation to the efficacy of adoptive NK cell therapy due to several immunosuppressive cytokines. TGF-beta, produced in excess by tumor cells, regulatory T cells and stromal cells, facilitates epithelial to mesenchymal transformation thereby promoting metastasis and fibrosis. This high TGF-beta milieu in cancer patients also impairs innate natural killer (NK) cell mediated cancer immunity through various mechanisms. Adoptive transfer of healthy donor NK cells will have limited clinical efficacy when highly activated NK cells are introduced into the immunosuppressive TGF-beta rich tumor microenvironment of cancer patients. We are testing several small molecule inhibitors of the TGF-beta receptor in combination with healthy donor NK cells with a goal of enhancing adoptive NK cell therapy in various disease models. Methods: Human NK cells isolated from healthy donor peripheral blood were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540). Cell culture media was supplemented with IL-2 (50mU/mL). For in vitro assays, at the beginning of expansion week 3 the culture conditions for NK cells was continued unchanged, supplemented with TGF-beta 1 ligand (TGF-B1) at 5ng/mL or 10ng/mL either alone or in combination with EW7197 (a TGF-beta Type 1 receptor inhibitor) or with EW7197 alone. These NK cells were tested with Calcein-AM release cytotoxicity assays at various time points from addition of TGF-B1 (0h, 36h, 72h and 96h). Briefly, NK cells were co-cultured with OCI-AML cells labeled with Calcein-AM at a ratio of 5NK:1 OCI-AML. At the end of 4 hours co-incubation, cytotoxicity was measured by relative fluorescence of calcein release into the culture supernatant compared with Triton-X induced complete target cell lysis. In a murine liver metastases model using the colon cancer cell line HCT116, 105 HCT116 cells were surgically implanted into NSG mouse spleens (following hemi-splenectomy). Mice in 2 groups (3/group) subsequently received 5 x 106 NK cells each (tail vein), weekly for two weeks, starting 10 days post-op. Another control group received vehicle infusions alone while a fourth control group received the TGF-beta inhibitor LY2157299 by oral gavage twice daily at 75mg/kg for two weeks. One group of mice receiving NK cells also received twice daily LY2157299 for two weeks starting with the first NK cell infusion. Mice receiving NK cells also received IL2 (75,000U IP) three times a week for two weeks. Results: At TGF-beta levels similar to that found in AML patients (5ng/ml), NK cell killing of OCI cells was markedly impaired progressively from 36h to 96h exposure. This reduced cytotoxic activity correlated with a 3-4 fold decrease in expression of NKG2D most marked at 96h(Figure 1). CD16 expression was also significantly reduced by 2-3 fold on TGF-beta exposed NK cells after 96h. The TGF-beta inhibitor EW7197 maintained NK cell killing as well as NKG2D and CD16 expression in the presence of TGF-B1 (Figure 1). At 32 days post-splenic implantation, vehicle mice (those that received HCT116 either alone or in combination with LY2157299 alone) appeared moribund and had grossly distended abdomens with ascites. All mice were autopsied at this time point. All control mice had grossly enlarged livers with significant metastases and absence of viable liver tissue (Fig 2). In contrast, mice treated with two weekly infusions of NK cells and LY2157299 for 4 weeks had no ascites and predominantly healthy livers. All mice that received NK cells alone had some evidence of liver metastases with various degrees of disease burden. Composite H&E stains of liver sections from 3 mice/group showed Discussion: Our preliminary results indicate that TGF-beta inhibition in combination with adoptive NK cell therapy can mitigate the immunosuppressive tumor microenvironment conferred by TGF-beta signaling. We are in the process of validating this approach in other preclinical models of both hematologic malignancies and solid tumors. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a devastating disease with a poor overall 5-yr survival rate and an antiquated and poorly tolerated therapy regimen. Despite an increasing knowledge of the molecular drivers of AML, new therapeutic targets are needed. GSK3β is a key signaling molecule that plays a role in several key oncogenic pathways including the β-catenin and NF-κB pathways, key pathways in AML and other cancers. Upregulation of GSK3β is correlated with poor prognosis in several cancers. We and others have shown GSK3β inhibition promotes AML differentiation as well as death of AML cells while sparing normal cells, suggesting GSK3β plays an important role in AML. Here we show by western blotting and confocal microscopy that GSK3β is upregulated and more nuclear in AML cells. We utilize flow cytometry to show AML cells express elevated GSK3β compared to normal marrow lineages (Average upregulation = 6 fold). We transform mouse hematopoietic progenitor cells with the MLL-AF9 oncogene and observe GSK3β upregulation as a course of AML leukemogenesis. These data suggest that elevated and nuclear GSK3β is characteristic of AML. To probe biological effects of GSK3β, we knocked down endogenous GSK3β and utilize a targetted, tetracycline-inducible GSK3β rescue model to show nuclear GSK3β specifically can promote AML growth. Nuclear GSK3β increases colony formation of OCI-AML3 and HL60 cell lines more potently than cytoplasmic (cyto) GSK3β in the same lines (OCI-AML3- Nuclear: 118% Cyto: 93% of control colony growth HL60- Nuclear: 117% Cyto: 100% of control colony growth, Nuclear difference- p At a molecular level, when compared to cells expressing cytoplasmic GSK3β, nuclear GSK3β more potently promotes NF-κB DNA binding and NF-κB mediated transcription as measured by EMSA assay and luciferase assay (Nuclear- 18 fold increase Cyto- 3 fold increase). Nuclear GSK3β more potently promotes activation of the NF-κB subunit p65, as measured by pS536, than cytoplasmic GSK3β. Nuclear GSK3β also promotes nuclear localization of p65. These data suggest that nuclear GSK3β can promote NF-κB activation in AML. Finally, we utilize imaging cytometry to show GSK3β is upregulated and more nuclear in clinical AML patient samples (N = 80) compared to normal bone marrow controls (N = 5). We also show nuclear localization of GSK3β and p65 correlate linearly in AML patient samples (N = 80 R2 = 0.44721 P Citation Format: James J. Ignatz-Hoover, Nathan Mackowski, David N. Wald. Elevated nuclear GSK3β promotes more aggressive disease in AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1154.

Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.