Cigarette smoke (CS) is the most common cause of chronic obstructive pulmonary diseases (COPD), including emphysema. CS exposure impacts all cell types within the airways and lung parenchyma, causing alveolar tissue destruction through four mechanisms: (1) oxidative stress; (2) inflammation; (3) proteaseinduced degradation of the extracellular matrix; and (4) enhanced alveolar epithelial and endothelial cell (EC) apoptosis. Studies in human pulmonary ECs demonstrate that macrophage migration inhibitory factor (MIF) antagonizes CS-induced apoptosis. Here, we used human microvascular ECs, an animal model of emphysema (mice challenged with chronic CS), and patient serum samples to address both the capacity of CS to alter MIF expression and the effects of MIF on disease severity. We demonstrate significantly reduced serum MIF levels in patients with COPD. In the murine model, chronic CS exposure resulted in decreased MIF mRNA and protein expression in the intact lung. MIF deficiency (Mif 2/2 ) potentiated the toxicity of CS exposure in vivo via increased apoptosis of ECs, resulting in enhanced CS-induced tissue remodeling. This was linked to MIF's capacity to protect against double-stranded DNA damage and suppress p53 expression. Taken together, MIF appears to antagonize CS-induced toxicity in the lung and resultant emphysematous tissue remodeling by suppressing EC DNA damage and controlling p53-mediated apoptosis, highlighting a critical role of MIF in EC homeostasis within the lung.

Cigarette smoke exposure is unquestionably the most frequent cause of chronic obstructive pulmonary disease (COPD) typified by emphysema. As the fifth leading cause of death worldwide, COPD is responsible for an estimated 2.5 million deaths annually. Accelerated endothelial cell apoptosis is now recognized as an important early determinant of lung destruction and resultant emphysema in COPD. As such, apoptosis becomes an attractive target for therapeutic intervention. However, the molecular determinants of cigarette induced endothelial cell apoptosis are incompletely defined. Recently, p53 protein expression has been demonstrated to be significantly higher in lung tissue from patients with emphysema controls suggesting the potential for a direct relationship between p53 expression and development of emphysema. Animal studies provide pre-clinical evidence that chronic cigarette smoke exposure is sufficient to increase p53 protein expression within the murine lung. Additional clinical data linking altered p53 expression with the risk of developing of emphysema comes from genetic studies which have linked polymorphisms of p53 to increased protein expression with an enhanced risk of developing COPD. Despite the abundance of data implicating apoptosis causally with development of emphysema, and data demonstrating changes in p53 expression during cigarette smoke-induced emphysema, a causal link between p53 and cigarette smoke-induced apoptosis is currently lacking. Here, we have shown that p53 is induced in HPEC exposed to CSE. Using the p53 inhibitor, pifithrin alpha (PFTa), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function/expression is required for CSE-mediated apoptosis in vitro. The expression of macrophage migration inhibitory factor (MIF), an anti-apoptotic cytokine produced by HPEC, also increases in response to CSE exposure. Addition of recombinant human MIF (rhMIF) prevents cell death from CSE exposure. Further, suppression of MIF or its receptor/binding partner, Jab-1, with RNAi enhances the sensitivity of HPEC to CSE via a p53-dependent pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via antagonism of p53-mediated apoptosis. We predict that genetic variants in the MIF promoter associated with altered MIF expression will impact clinical responses to chronic cigarette smoke in patients. This work supports a potential causal link between increased p53 expression and the increased apoptosis of emphysema and implicates altered MIF expression or function as a potential determinant of disease severity.

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Exposure to cigarette smoke (CS) is the most common cause of emphysema, a debilitating pulmonary disease histopathologically characterized by the irreversible destruction of lung architecture. Mounting evidence links enhanced endothelial apoptosis causally to the development of emphysema. However, the molecular determinants of human endothelial cell apoptosis and survival in response to CS are not fully defined. Such determinants could represent clinically relevant targets for intervention. We show here that CS extract (CSE) triggers the death of human pulmonary macrovascular endothelial cells (HPAECs) through a caspase 9-dependent apoptotic pathway. Exposure to CSE results in the increased expression of p53 in HPAECs. Using the p53 inhibitor, pifithrin-a (PFT-a), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function and expression are required for CSE-mediated apoptosis. The expression of macrophage migration inhibitory factor (MIF), an antiapoptotic cytokine produced by HPAECs, also increases in response to CSE exposure. The addition of recombinant human MIF prevents cell death from exposure to CSE. Further, the suppression of MIF or its receptor/binding partner, Jun activation domain-binding protein 1 (Jab-1), with RNAi enhances the sensitivity of human pulmonary endothelial cells to CSE via a p53-dependent (PFT-a-inhibitable) pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via the antagonism of p53-mediated apoptosis.

Background: The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL), with expression of the delta isoform of PI3K largely restricted to lymphocytes. Clinical evaluation of PI3K-δ inhibitors, such as idelalisib, in CLL patients has been promising. CLL patients with 17p deletion (17pdel) have displayed inferior responses to chemo-immunotherapy, and we hypothesized that inhibiting the PI3K/AKT pathway would be equally efficacious in patients with and without 17pdel. TGR-1202 is a novel PI3K-δ specific inhibitor with unique pharmacologic properties and demonstrated in-vitro activity. Herein, we evaluate the in vitro effect of TGR-1202 on CLL lymphocytes, specifically evaluating differences between 17pdel CLL and non-17pdel CLL samples. Methods: We collected blood from CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers. CLL lymphocytes were isolated using negative selection yielding greater than 95% purity. Primary CLL cells were incubated with serial dilutions of TGR-1202 tested for apoptosis by activated caspase-3 and 7AAD staining measured by flow cytometry. Cytotoxicity was measured using the colorimetric MTS reagent. Phosphorylated AKT (S473) was measured by flow cytometry after incubation with TGR-1202 followed by incubation with anti-IgM or anti-IgD. AKT phosphorylation was quantified by median fluorescent intensity (MFI). Results: We have evaluated TGR-1202 in 15 CLL lymphocyte samples, 10 with normal non-17pdel cytogenetics, and 5 with 17pdel. TGR-1202 induced cytotoxicity and apoptosis in a dose-dependent manner in concentrations between 4 nM and 25 μM with no significant difference in results observed between normal CLL lymphocytes those that had 17pdel cytogenetics. The mean ED50 in 17pdel CLL cells was 326 nM, whereas the mean ED50 in non-17pdel CLL cells was 887 nM, although due to the small sample size, this difference was not statistically significant (p = 0.16, t-test). We also found that TGR-1202 significantly suppressed AKT phosphorylation in CLL lymphocytes. Conclusions: TGR-1202 is a potent PI3K-δ inhibitor that suppresses AKT phosphorylation and induces apoptosis-dependent cytotoxicity in primary CLL lymphocytes, both with favorable and adverse cytogenetics. We observed that higher concentrations of TGR-1202 were required to induce cytotoxicity in non-17pdel CLL lymphocytes on average, than in 17pdel CLL. Evaluation of additional CLL cell samples with 17pdel is ongoing to confirm initial findings. A Phase I trial with once-daily TGR-1202 is currently ongoing in patients with hematologic malignancies, including CLL. To date, TGR-1202 has been well tolerated, with no drug-related hepatic toxicities observed. Our initial observations suggest that clinical evaluation of TGR-1202 enriched in CLL patients with 17pdel may be warranted. Citation Format: Daphne R. Friedman, Tiffany Simms, Sallie D. Allgood, Danielle M. Brander, Peter Sportelli, Hari P. Miskin, Swaroop Vakkalanka, Srikant Viswanadha, J. Brice Weinberg, Mark C. Lanasa. The PI3K-δ inhibitor TGR-1202 induces cytotoxicity and inhibits phosphorylation of AKT in 17p deleted and non-17p deleted CLL cells in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4518. doi:10.1158/1538-7445.AM2014-4518

Introduction Vitamin D deficiency is common in the general population. Approximately 25-50% of adult patients seen at routine visits in the United States are found to have an insufficient vitamin D level. Vitamin D has been shown to be prognostic in several types of cancers including breast, prostate and colon cancer. Vitamin D activates a nuclear transcription factor that regulates the expression of almost 200 genes which modulate a variety of cellular processes including angiogenesis, differentiation, proliferation, and apoptosis. Recent research has shown that vitamin D levels may have a prognostic effect in patients with chronic lymphocytic leukemia (CLL), where 25-OH vitamin D insufficiency was associated with shortened time to treatment and poorer overall survival. A centrally important unanswered question relates to causation: does vitamin D insufficiency yield more aggressive cancer disease biology, or do intrinsically progressive cases of CLL cause vitamin D insufficiency? We hypothesized that vitamin D insufficiency alters CLL cell biology and favors a more aggressive disease phenotype. Methods Untreated patients within 12 months of initial diagnosis of CLL from Duke University Hospital and the Durham VA were studied. Serum samples from 185 patients were assayed for the 25-OH vitamin D level (immunochemiluminometric assay). A multivariate analysis was performed using: age, race, gender, Rai stage, CD38, Zap70, hierarchical FISH, IGHV, and season of diagnosis to determine whether vitamin D levels are a significant predictor of OS and TTT in this group. Global mRNA expression from 23 patients was analyzed using Affymetrix U133 Plus 2.0 arrays as a function of vitamin D level and gene list generated for those with p values < 10-5. rtPCR was performed on samples from an additional 50 patients to validate the findings from the mRNA expression analysis. Linear regression analysis was conducted to evaluate for significant associations between genes and 25-OH vitamin D levels. An in vitro assessment of 1,25-di-OH vitamin D effects on CLL cell viability in serum free media was evaluated using an MTS assay. Results The mean vitamin D level amongst the group of 185 patients was 25.6± 9.7 ng/mL. Eighty-nine patients had a vitamin D level less than 25 and 96 had a level above 25, which we used as our cutoff, as prior reports have used this level to define insufficiency in CLL. Thirty-one of 95 (33%) of the sufficient vitamin D group were treated versus 39 of 89 (44%) of the insufficient vitamin D group (p=0.12). Among those requiring treatment, the mean TTT was approximately the same between the two groups: 4.7±0.3 yrs for the higher vitamin D group vs. 4.6±0.4 yrs for the insufficient group (p=0.126). OS for the higher vitamin D group was 8.3±0.3 vs. 7.0±0.2 years for the lower vitamin D group (p=0.935). Multivariable analysis showed that IGHV mutation (HR = 0.386; p=0.0159) and Rai stage 0 or 1 (HR = 0.174; p=0.0002) predicted TTT, while age and race influenced OS, with age>62 conferring greater risk of death (p=0.0191) and African Americans having decreased survival (p=0.0110). Preliminary studies of gene expression data identified eight probes that were differentially expressed as a function of vitamin D level. rtPCR was then performed on GPR82, MPZL3, FBXW4, ROR1, and CXCL11 to validate these results. Linear regression confirmed that ROR1 and FBXW4 gene expression correlated with vitamin D level (p=0.0065; r2=0.144 and p=0.0185; r2=0.110, respectively). High levels of ROR1 are observed in B-CLL. FBXW4 has been shown to be mutated or under-expressed in a variety of human cancer cell lines. Early in vitro cytotoxicity of 1,25 di-OH vitamin D in CLL (n = 5 patient derived samples) showed an IC50 = 334 nM. Discussion Our results show that the basal level of vitamin D is not significantly correlated with either OS or TTT in CLL in contrast to previous studies. No interaction between vitamin D levels and race, age, gender, Rai stage, IGHV mutation, season of diagnosis was observed. However, ongoing in vitro experiments show that 1,25 vitamin D is cytotoxic to CLL, raising the intriguing possibility that intermittent bolus dosing could potentially be used therapeutically. Further, we have identified specific genes where quantitative gene expression is correlated with basal vitamin D levels. These findings expand our understanding of the interaction between vitamin D and B cell malignancies. Disclosures: No relevant conflicts of interest to declare.

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Respiratory syncytial virus (RSV) infection is the most common cause of lower respiratory tract infection in infants and elderly adults. Previous studies in humans and in animal models suggest that early infection with RSV may predispose to the development of asthma. However, the molecular mechanisms by which RSV induces changes in the immuno-environment of the airway are still largely unknown. The purpose of the present research was to identify the molecular effectors involved in the host response to RSV infection in alveolar macrophages (AMs). Seven day old mice were infected with RSV (4 x 104 TCID50/g body weight). Total RNA was isolated from airway AMs 5 hours post-infection and processed to perform expression analysis using the Affymetrix GeneChip (Mouse Genome 430 2.0s). A total of 424 genes were observed to be differently expressed. Of these, 187 genes were found to be up-regulated and 237 genes to be down-regulated. Many of these genes take part in cell signaling pathways including the IL-6 and p38 MAPK pathways. Interestingly, 10 genes were found to be involved in apoptosis. RSV infection resulted in complex and immediate cellular responses in AMs. Preliminary data suggests that RSV inhibits apoptosis of AMs. IL-6, amphiregulin and matrix metalloproteinase-13 may play roles in the subsequent development of asthma. This publication was made possible by NIH grant number P20 RR020159 from the LSU/Tulane COBRE-CEIDR Program of the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.