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Purpose: Patients with advanced non–small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype. Experimental Design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens. Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell–mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells. Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

IL-6 modulates expression of NK receptor and their ligands in EGFR mutant tumors.

PTEN loss in melanoma promotes resistance to T cell-mediated anti-tumor activity in the context of concurrent a selective inhibitor for mutant BRAF.

Supplementary Table S1. Patient characteristics of the single-agent anti-PD-1 cohort. Supplementary Table S2. List of primers used for gene expression analysis by real-time PCR. Supplementary Table S3. List of autophagy-related genes for overexpressing experiment.

PDF file, 177K, Uveal melanoma cells were treated with increasing doses of MEKi or PI3Ki and impedance measurements ascertained at 10 min intervals.

PDF file, 78K, Cell death assessment following combination MEKi + PI3Ki treatment in BIM siRNA targeted uveal melanoma cells.

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PDF file, 106K, Uveal melanoma cells (UPMD1 and OMM1) were treated with increasing concentrations of MEKi (0 - 1000 nM) or PI3Ki (0 - 1000 nM) x 4hours. Western blot analysis of phosphorylated MAPK1/2, total MAPK2, phosphorylated AKT, and total AKT following treatment are presented.

PDF file, 55K, Baseline protein expression of phosphorylated MAPK1/2, total MAPK1/2, phosphorylated AKT, total AKT in uveal melanoma cell lines.

Purpose: Activating Q209L/P mutations in GNAQ or GNA11 (GNAQ/11) are present in approximately 80% of uveal melanomas. Mutant GNAQ/11 are not currently therapeutically targetable. Inhibiting key down-stream effectors of GNAQ/11 represents a rational therapeutic approach for uveal melanomas that harbor these mutations. The mitogen-activated protein/extracellular signal–regulated kinase/mitogen-activated protein kinase (MEK/MAPK) and PI3K/AKT pathways are activated in uveal melanoma. In this study, we test the effect of the clinically relevant small molecule inhibitors GSK1120212 (MEK inhibitor) and GSK2126458 (pan class I PI3K inhibitor) on uveal melanoma cells with different GNAQ/11 mutation backgrounds.Experimental Design: We use the largest set of genetically annotated uveal melanoma cell lines to date to carry out in vitro cellular signaling, cell-cycle regulation, growth, and apoptosis analyses. RNA interference and small molecule MEK and/or PI3K inhibitor treatment were used to determine the dependency of uveal melanoma cells with different GNAQ/11 mutation backgrounds on MEK/MAPK and/or PI3K/AKT signaling. Proteomic network analysis was done to unveil signaling alterations in response to MEK and/or PI3K small molecule inhibition.Results:GNAQ/11 mutation status was not a determinant of whether cells would undergo cell-cycle arrest or growth inhibition to MEK and/or phosphoinositide 3-kinase (PI3K) inhibition. A reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK + PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant–dependent manner.Conclusions: MEK + PI3K inhibition may be an effective combination therapy in uveal melanoma, given the inherent reciprocal activation of these pathways within these cells. Clin Cancer Res; 18(16); 4345–55. ©2012 AACR.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Targeted agents, such as EGFR tyrosine kinase inhibitors (TKIs) and KRAS G12C inhibitors, have provided clinical benefits for patients and can yield significant tumor shrinkage. Inevitably, however, there remain residual tumors that harbor drug-tolerant persister cells (DTPCs) not killed by initial treatment, leading to emerging of drug-resistant cells (DRCs) and tumor recurrence. The identification of effective treatment strategies to target DTPCs is a critical and unmet need. We hypothesized that DTPCs can be targeted using chimeric antigen receptor (CAR)-based cellular therapy approaches directed at cell surface proteins expressed on DTPCs and that the combination of such approaches with targeted agents will more effectively delay the emergence of fully drug-resistant cells than targeted agents alone. To this end, we first performed luciferase-based killing assays and observed that EGFR-targeting CAR-T and CAR-NK cells displayed potent cytotoxicity against DTPCs of osimertinib-treated EGFR mutant NSCLC cells. Moreover, osimertinib DTPCs showed increased sensitivity to EGFR CAR-T and CAR-NK cells compared to their parental cells, likely due to the upregulation of EGFR on the cell surface following osimertinib treatment. In a patient-derived xenograft model of HER2YVMA mutant lung cancer, we observed that HER2 CAR-NK cells showed enhanced antitumor activity in mice treated with the HER2 inhibitor, poziotinib, for two weeks, compared to CAR-NK cells only, poziotinib only, or mice treated upfront with HER2 CAR-NK cells plus poziotinib. These in vitro and in vivo data support that CAR-based cellular therapy can effectively eliminate DTPCs. To identify additional candidate cell surface proteins expressed on DTPCs that could be targeted by CAR-based cell therapy approaches, we interrogated RNA expression from our panel of DTPCs as well as public datasets containing DTPCs of targeted agents. Likewise, we investigated the expression of cell surface proteins using datasets of parental and DRCs to identify the common candidate targets that could be exploited on both DTPCs and DRCs. Our analysis identified CD70 as being consistently upregulated in both EGFR TKI-resistant cells and DTPCs of osimertinib-treated EGFR mutant NSCLC. Moreover, we observed CD70 upregulation in DTPCs and DRCs of other targeted agents, including KRAS, ALK, and RET inhibitors, suggesting that CD70 may be a common target in DTPCs across oncogenic drivers. Furthermore, we determined that CD70-targeting CAR-T and CAR-NK cells showed promising in vitro activity against the DTPCs of osimertinib-treated EGFR mutant NSCLC. These results demonstrate CAR-based cellular therapy as an effective approach to target DTPCs and identify CD70 as a novel therapeutic target for combatting DTPCs in NSCLC. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Xiaoxing Yu, Alissa Poteete, Qian Huang, Simon Heeke, John Heymach. CD70-targeting CAR-T and CAR-NK cells demonstrate potent activity against NSCLC drug-tolerant persister cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 891.

Activating ERBB2 (HER2) mutations occur across multiple malignancies, and although mutational hotpots vary by disease, HER2 mutations most frequently occur in the tyrosine kinase domain. Among lung cancer patients, the most common HER2 mutation is the exon 20 insertion mutations, Y772dupYVMA, which accounts for over 30% of all HER2 mutations in lung cancer. Recent clinical testing of trastuzumab deruxtecan (T-DXd), a HER2 antibody-drug conjugate (ADC), against HER2 mutant non-small cell lung cancer (NSCLC) demonstrated a confirmed objective response of 55%, a median duration of response of 9.3 months, and a median overall survival of 17.8 months, which ultimately led to the recent approval of T-DXd for HER2 mutant NSCLC. Acquired resistance to targeted agents remains a major clinical challenge, and HER2-mutant patients that initially respond to T-DXd will eventually develop progressive disease. Thus, understanding mechanism of T-DXd resistance and identifying targeted agents that remain effective in cells with acquired T-DXd resistance is of great clinical importance. We generated Ba/F3 cells expressing the HER2 YVMA mutation and assessed sensitivity to HER2 targeting agents by Cell Titer Glo assay. HER2 YVMA cells were sensitive to the EGFR/HER2 tyrosine kinase inhibitor (TKI) poziotinib and the HER2 ADC T-DXd. To generate HER2 ADC resistant cells, we cultured Ba/F3 HER2 YVMA cells in T-DXd continuously for 8 weeks at which point cells resumed rapid proliferation. Cell Titer Glo assay confirmed that Ba/F3 HER2 YVMA cells were sensitive to T-DXd with an IC50 of 195 μg/ml, whereas Ba/F3 HER2 YVMA T-DXd-resistant cells had an IC50 of greater than 1000 μg/ml. Notably, T-DXd resistant cells remained highly sensitive to the HER2 TKI poziotinib with an IC50 of 14 nM and 7.5 nM for T-DXd-resistant and parental cells, respectively. Moreover, T-DXd-resistant cells demonstrated reduced sensitivity to the topoisomerase inhibitor topotecan with an IC50 of 200 nM whereas parental cells had an IC50 of 66 nM, suggesting that acquired T-DXd resistance may be mediated by loss of sensitivity to the ADC payload. Collectively, these data demonstrate that HER2 TKIs such as poziotinib may retain anti-tumor cell activity in HER2 mutant tumor cells with acquired resistance to HER2 ADCs. Citation Format: Monique B. Nilsson, Alissa Poteete, Hibiki Udagawa, Xiaoxing Yu, Junqin He, Xiuning Le, John Heymach. Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 383.

Reduced mitochondrial ATP synthase (ATPS) capacity plays crucial roles in the pathogenesis of metabolic disorders. However, there is currently no effective strategy for synchronously stimulating the expressions of ATPS key subunits to restore its assembly. This study determined the roles of mitochondrial protein FAM3A in regulating the activity and assembly of ATPS in hepatocytes. FAM3A is localized in mitochondrial matrix, where it interacts with F1-ATPS to initially activate ATP synthesis and release, and released ATP further activates P2 receptor-Akt-CREB pathway to induce FOXD3 expression. FOXD3 synchronously stimulates the transcriptions of ATPS key subunits and assembly genes to increase its assembly and capacity, augmenting ATP synthesis and inhibiting ROS production. FAM3A, FOXD3 and ATPS expressions were reduced in livers of diabetic mice and NAFLD patients. FOXD3 expression, ATPS capacity and ATP content were reduced in various tissues of FAM3A-deficient mice with dysregulated glucose and lipid metabolism. Hepatic FOXD3 activation increased ATPS assembly to ameliorate dysregulated glucose and lipid metabolism in obese mice. Hepatic FOXD3 inhibition or knockout reduced ATPS capacity to aggravate HFD-induced hyperglycemia and steatosis. In conclusion, FAM3A is an active ATPS component, and regulates its activity and assembly by activating FOXD3. Activating FAM3A-FOXD3 axis represents a viable strategy for restoring ATPS assembly to treat metabolic disorders.

Genetically modified (GM) crops expressing insecticidal and herbicide-tolerant traits provide a new approach to agriculture production, but concerns about food safety were often raised by the public. The present research shows the findings of the nutritional assessment of ZDAB3 expressing insecticidal Cry proteins (Cry1Ab and Cry2Ab) and EPSPS protein (G10evo-EPSPS). The key nutrients and anti-nutrients of ZDAB3 maize were examined and contrasted with those of its non-transgenic control maize grown at the same locations during three planting seasons. The values for proximates, amino acids, fatty acids, minerals, vitamins, phytic acid, and trypsin inhibitor assessed for ZDAB3 were comparable to those of its non-transgenic control maize or within the range of values reported for other commercial lines. In addition, no adverse effects related to the G10evo-EPSPS protein in mammals were observed. These data indicated that the expression of Cry1Ab, Cry2Ab, and G10evo-EPSPS proteins in ZDAB3 maize does not affect the nutritional compositions, and ZDAB3 maize is equivalent to non-transgenic maize regarding those important compositions.

Mitochondrial FAM3A has been revealed to be a viable target for treating diabetes and nonalcoholic fatty liver disease (NAFLD). However, its distinct mechanism in ameliorating hepatic steatosis remained unrevealed. High-throughput RNA sequencing revealed that carnitine palmityl transferase 2 (CPT2), one of the key enzymes for lipid oxidation, is the downstream molecule of FAM3A signaling pathway in hepatocytes. Intensive study demonstrated that FAM3A-induced ATP release activated P2 receptor to promote the translocation of calmodulin (CaM) from cytoplasm into nucleus, where it functioned as a co-activator of forkhead box protein A2 (FOXA2) to promote the transcription of CPT2, increasing free fatty acid oxidation and reducing lipid deposition in hepatocytes. Furthermore, antidepressant imipramine activated FAM3A-ATP-P2 receptor-CaM-FOXA2-CPT2 pathway to reduce lipid deposition in hepatocytes. In FAM3A-deficient hepatocytes, imipramine failed to activate CaM-FOXA2-CPT2 axis to increase lipid oxidation. Imipramine administration significantly ameliorated hepatic steatosis, hyperglycemia and obesity of obese mice mainly by activating FAM3A-ATP-CaM-FOXA2-CPT2 pathway in liver and thermogenesis in brown adipose tissue (BAT). In FAM3A-deficient mice fed on high-fat-diet, imipramine treatment failed to correct the dysregulated lipid and glucose metabolism, and activate thermogenesis in BAT. In conclusion, imipramine activates FAM3A-ATP-CaM-FOXA2-CPT2 pathway to ameliorate steatosis. For depressive patients complicated with metabolic disorders, imipramine may be recommended in priority as antidepressive drug.

Approximately 15% of all patients with non-small cell lung cancer (NSCLC) and nearly 35% of Asian patients with NSCLC harbor activating mutations within the epidermal growth factor receptor (EGFR). Although these patients are initially highly sensitive to first or second generation EGFR tyrosine kinase inhibitors (TKIs) including erlotinib or third-generation inhibitors including osimertinib, EGFR TKI-refractory disease inevitably emerges. While therapeutic strategies to target resistant disease that emerges though secondary EGFR mutations or MET amplification have been developed, there remains a void of therapeutic options for patients where resistance occurs through EGFR-independent mechanism such as epithelial to mesenchymal transition (EMT) or transformation to small cell lung cancer (SCLC). To identify cell surface proteins that could be targeted by antibody-based or adoptive cell therapy approaches we interrogated RNAseq data from EGFR mutant NSCLC cell lines (HCC827 and HCC4006) and their associated EGFR TKI resistant variants previously shown to have developed resistance through EMT and filtered gene expression data to include only genes which transcribed proteins localized to the cell surface. We identified CD70 as a being highly upregulated in EGFR TKI resistant cells (p = 7.2e-42). Given that CD70 expression is highly restricted and only transiently expressed on immune cells, CD70 was selected as a top candidate cell surface protein for targeting studies. Western blotting and flow cytometry analysis confirmed CD70 protein levels to be highly upregulated in EGFR TKI resistant cells that had undergone EMT but not in cells harboring secondary EGFR mutations or MET amplifications. We also observed CD70 upregulation in osimertinib-treated drug tolerant persister cells, indicating that CD70 upregulation is an early event in the evolution of TKI resistance. Moreover, patient-derived models of acquired EGFR TKI resistance also exhibited CD70 positivity. Our data also indicated that in EGFR mutant NSCLC cells, CD70 could be upregulated through decreased CD70 promoter methylation as well as by the EMT regulators, transforming growth factor-β (TGF-β) and ZEB1, both of which were upregulated in TKI resistant cells. In EGFR TKI resistant cells, CD70 knockdown impaired cell viability and invasiveness, and stimulation of CD70 using the exogenous binding partner CD27 resulted in activation of AKT and MAPK, pathways known to be re-activated with acquired TKI resistance. CD70-targeting approaches including anti-CD70 antibody drug conjugates (ADCs) and CD70-targeting CAR T cell and CAR NK cells showed promising in vitro and in vivo activity against CD70 positive tumor cells and in osimertinib drug-tolerant persister cells. These results identify CD70 as a novel therapeutic target for EGFR mutant tumors with acquired EGFR TKI resistance that merits further investigation in the clinic. Citation Format: Monique B. Nilsson, Yan Yang, Sonia Patel, Simon Heeke, Xiuning Le, Thiru Aruguman, Jacqulyne Robichaux, Xiaoxing Yu, Alissa Poteete, Xiaoyang Ren, Lixia Diao, Li Shen, Qi Wang, Fahao Zhang, Leticia Campos Clemente, Luisa Solis Soto, Chunhua Shi, Hai Tran, Jason Bock, Jing Wang, Ignacio I. Wistuba, John D. Minna, John V. Heymach. CD70 is a novel therapeutic target for EGFR mutant NSCLC with acquired, EMT-associated EGFR TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1827.

Around 10-15% of non-small cell lung cancer (NSCLC) cases in the United States harbor an activating mutation in epidermal growth factor receptor (EGFR) which are initially highly sensitive to treatment EGFR tyrosine kinase inhibitors (TKI) before eventually developing resistance to these agents. Therefore, there is a current unmet clinical need to identify novel treatment options for this patent population. While anti-PD-1/PD-L1 immunotherapy has been effective for many NSCLC patients, EGFR-mutant tumors have response rates of less than 10%. The mechanisms driving resistance to immunotherapy in EGFR-mutant NSCLC are not well understood. We previously reported that IL-6 is highly upregulated in NSCLC cells with acquired resistance to EGFR-TKIs. Because IL-6 is a pleiotropic cytokine known to impact immune populations within the tumor microenvironment, we hypothesized that IL-6 may drive immunosuppression in EGFR mutant NSCLC. Using genetically engineered mouse models (GEMMs) of EGFR-mutant NSCLC with and without IL-6 knockout, we evaluated the effects of IL-6 on the tumor microenvironment. EGFR-mutant mice with knockout of IL-6 had a significantly increased overall survival compared to those with intact-IL-6. Knockout of IL-6 increased total immune infiltration in these tumors as assessed by flow cytometry. Infiltrating T cells from IL-6 knockout mice displayed a smaller population of T helper 17 cells compared to IL-6-expressing tumors. Knockout of IL-6 also increased the population of infiltrating activated CD8 T cells and a reduced T-regulatory cell population. Due to these changes in T cell activity, we wanted to determine any potential synergistic effects of IL-6 blockade used during anti-PD-1 therapy. We observed that combination treatment extended survival in EGFR-mutant NSCLC mice. Corresponding in vitro T cell cytotoxicity assays confirmed that EGFR-TKI resistant cells treated with IL-6 blocking antibody were more sensitive to T cell-mediated killing. Furthermore, infiltration of NK cells was increased in tumors with knockout of IL-6. Infiltrating NK cells found in these IL-6 knockout mice displayed a more activated phenotype as demonstrated by increased expression of NKG2D. Next, we evaluated the effects of IL-6 from conditioned media collected from human EGFR-mutant and TKI-resistant cells lines on human T and NK cells cultured ex vivo. e determined that IL-6 suppressed expression of activation markers including granzyme B and IFN-y on the surface of both T and NK cells. Blockade of IL-6 increased NK-mediated cytotoxic killing of EGFR-TKI resistant cells in vitro. In conclusion, our data indicates that in EGFR-TKI resistance, upregulated IL-6 suppresses T and NK cell cytotoxic potential and drives immunosuppression. Citation Format: Sonia Patel, Monique Nilsson, Yan Yang, Xiaoxing Yu, Fahao Zhang, Alissa Poteete, Xiaoyang Ren, Li Shen, Jing Wang, Xiuning Le, John Heymach. IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3125.

The leading cause of cancer-related deaths in the US is non-small cell lung cancer (NSCLC), and about 10-15% of NSCLC cases harbor EGFR activating mutations. While these patients are initially responsive to EGFR tyrosine kinase inhibitors (TKIs), they eventually experience progression and develop acquired TKI resistance. Currently, there are no approved therapies for NSCLC patients with acquired resistance to the third-generation EGFR TKI osimertinib (OSI). Moreover, EGFR-TKI resistant NSCLCs are refractory to immune checkpoint inhibitors. Therefore, novel treatment strategies are urgently needed. Chimeric antigen receptors (CARs) have been used to enhance the anti-tumor activity of immune effector cells. Here, we developed CAR-based therapies targeting EGFR as a novel strategy to overcome immune suppression and target EGFR TKI-resistant NSCLC. We designed multi-generational EGFR CAR constructs and transduced them into T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. We then evaluated their activity against NSCLC cell lines in vitro using firefly luciferase assays. EGFR CAR-T and CAR-NK cells showed potent cytotoxicity against NSCLC cells expressing EGFR including EGFR wild-type and mutant NSCLCs, as well as NSCLC cells with acquired resistance to OSI. However, EGFR CAR-T cells showed decreased killing activity against OSI-resistant NSCLC cells compared to their parental cells. In contrast, EGFR CAR-NK cells showed stronger cytotoxicity against NSCLC with acquired OSI resistance as compared to parental cells. Transcriptomic analysis revealed that NSCLC cells with acquired resistance to TKI had undergone epithelial-mesenchymal transition and had downregulated expression of genes related to antigen presentation and upregulated expression of B7-H6 (NCR3LG1), which have been reported to be associated with increased responsiveness to NK cells. Moreover, treatment of TKI resistant cells with OSI resulted in upregulation of cell surface EGFR and potentiated CAR-NK cells-mediated killing. TGF-β is a critical immune-suppressive cytokine. We found that EGFR-TKI resistant NSCLC cells elaborated expression of TGF-β as compared to parental cells. Blockade of the TGF-β pathway using galunisertib significantly enhanced the activity of EGFR CAR-NK cells against OSI-resistant NSCLC cells. Next, we engineered the dominant-negative TGF-β receptor II (DNTGFBRII) into our EGFR CAR constructs. The expression of DNTGFBRII inhibited the phosphorylation of SMAD2 and the downregulation of granzyme B and perforin induced by TGF-β. DNTGFBRII-CAR-NK cells showed higher killing activity against OSI-resistant NSCLC cells and were resistant to TGF-β-mediated immunosuppression. In conclusion, EGFR CAR-NK cells show significant anti-tumor activity to EGFR TKI-resistant NSCLC cells, and the cytotoxicity is enhanced in combination with OSI treatment and blockade of the TGF-β pathway. Citation Format: Yan Yang, Monique Nilsson, Sonia Patel, Jacqulyne Robichaux, Alissa Poteete, Xiaoxing Yu, Fahao Zhang, Simon Heeke, Yu Qian, Junqin He, John Heymach. EGFR CAR-NK cells demonstrate potent activity against EGFR TKI resistant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 548.

Introduction The treatment of patients with EGFR-mutant NSCLC with vascular endothelial growth factor (VEGF) inhibitors in combination with EGFR inhibitors provides a greater benefit than EGFR inhibition alone, suggesting that EGFR mutation status may define a patient subgroup with greater benefit from VEGF blockade. The mechanisms driving this potentially enhanced VEGF dependence are unknown. Methods We analyzed the effect of EGFR inhibition on VEGF and HIF-1α in NSCLC models in vitro and in vivo. We determined the efficacy of VEGF inhibition in xenografts and analyzed the impact of acquired EGFR inhibitor resistance on VEGF and HIF-1α. Results NSCLC cells with EGFR-activating mutations exhibited altered regulation of VEGF compared with EGFR wild-type cells. In EGFR-mutant cells, EGFR, not hypoxia, was the dominant regulator of HIF-1α and VEGF. NSCLC tumor models bearing classical or exon 20 EGFR mutations were more sensitive to VEGF inhibition than EGFR wild-type tumors, and a combination of VEGF and EGFR inhibition delayed tumor progression. In models of acquired EGFR inhibitor resistance, whereas VEGF remained overexpressed, the hypoxia-independent expression of HIF-1α was delinked from EGFR signaling, and EGFR inhibition no longer diminished HIF-1α or VEGF expression. Conclusions In EGFR-mutant NSCLC, EGFR signaling is the dominant regulator of HIF-1α and VEGF in a hypoxia-independent manner, hijacking an important cellular response regulating tumor aggressiveness. Cells with acquired EGFR inhibitor resistance retained elevated expression of HIF-1α and VEGF, and the pathways were no longer EGFR-regulated. This supports VEGF targeting in EGFR-mutant tumors in the EGFR inhibitor–naive and refractory settings.

T cell–mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell–mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell–mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti–PD-1 and anti–CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K–AKT pathway inhibitors. Significance: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K–AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K–AKT pathway to increase the efficacy of immunotherapy. Cancer Discov; 6(2); 202–16. ©2015 AACR. See related commentary by Rizvi and Chan, p. 128. This article is highlighted in the In This Issue feature, p. 109

The purpose of this study was to determine the role of BAP1 levels in cutaneous melanoma (CM). BAP1 is a tumor suppressor in which loss of heterozygosity (LOH) from mutation and copy number alteration is well described in germline and somatic cancers. Although BAP1 genomic alterations in CM are extremely rare (2% of 665 samples from 5 datasets), marked variability in BAP1 expression is observed in CM. We show that low nuclear BAP1 levels portend a significantly worse clinical outcome in stage III CM (n=37, log rank p ≤0.01 for both overall survival and progression-free survival). Gene Set Enrichment Analysis (GSEA) revealed low BAP1 expression to be most highly ranked with an increased epithelial–mesenchymal transition (EMT) gene expression profile in CM tumors (n=379, FDR q = 1.34E-26) and cell lines (n=53, FDR q = 2.86E-116). We identify the expression of ZEB1, a master regulator of EMT, to be significantly associated with low BAP1 expression in CM tumors and cell lines (p= 1.5E-04 and 3.3E-05, respectively). Analysis of the BAP1 promoter indicates three canonical ZEB1 binding sites. Functional experiments show ZEB1 to bind to the BAP1 promoter, and luciferase activity assays indicate that ZEB1 acts as a transcriptional suppressor of BAP1 expression with differential utilization of the promoter binding sites. Targeted reduction of endogenous ZEB1 caused increased BAP1 levels, while targeted reduction of BAP1 did not modulate ZEB1 levels, consistent with ZEB1 having a suppressive effect on BAP1. Phenotypically, targeted reduction of BAP1 in CM cells resulted in a switch from a more differentiated, melanocytic state, to a less differentiated, more migratory and invasive phenotype. Extinguishing melanocyte-specific BAP1 in mice with a BRAF V600E mutant genetic background resulted in the emergence of primary melanoma tumors, with a marked EMT gene expression profile, and resultant metastases. Given the phenotypic changes associated with BAP1 levels in our mouse and human studies, we then tested the effect of modulating BAP1 on BRAF targeted therapy. Exogenous expression of BAP1 sensitized BRAF inhibitor (vemurafenib)-resistant melanoma cells, while the targeted reduction of BAP1 desensitized BRAF inhibitor-sensitive melanoma cells. BRAF mutant/BAP1 loss mice failed to exhibit a marked response to vemurafenib treatment compared to control mice. These data implicate regulation of BAP1 to be a major mechanism that characterizes a highly malignant and treatment-resistant subset of tumors. Our study indicates that nongenomic reduction in BAP1 through ZEB1 transcriptional modulation may be a key factor in aggressive CM. This abstract is also being presented as Poster A30. Citation Format: Haifeng Zhu, Junna Oba, Xiaoxing Yu, Caitlin A. Creasy, Marie-Andrée Forget, Fernando Carapeto, Cara L. Haymaker, Chang-Jiun Wu, Tatiana V. Karpinets, Wei-Lien Wang, Michael T. Tetzlaff, Alexander J. Lazar, Gordon B. Mills, Amanda R. Moore, Yu Chen, Jianhua Zhang, Jeffrey E. Gershenwald, Jennifer A. Wargo, Chantale Bernatchez, Patrick Hwu, P. Andrew Futreal, Scott E. Woodman. Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR03.

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation.

cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3′,5′-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25–30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5′-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr853, which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.

Background and Purpose Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets. Experimental Approach Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by elisa. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy. Key Results Incubation of INS-1E cells and rat islets with HG (30 mmol·L−1; 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m+ mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine. Conclusions and Implications Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine.

The down-regulation of α-adrenoceptor-mediated signaling casacade has been implicated in obesity but the underlying mechanism remains largely unknown. The present study investigated whether inositol 1,4,5-trisphosphate (IP3) receptor and protein kinase C (PKC) were involved in the reduction of α1-adrenoceptor agonist phenylephrine-evoked contraction in aortae of high fat diet-induced obese (DIO) mice. C57BL/6 mice were fed with a rodent diet containing 45 kcal% fat for 16 weeks to induce obesity. Isolated mouse aortae were suspended in myograph for isometric force measurement. Protein phosphorylations and expressions were determined by Western blotting. In C57BL/6 mouse aortae, phenylephrine-induced contraction was partially inhibited by either IP3 receptor antagonist heparin or PKC inhibitor GFX, and the combined treatment with heparin and GFX abolished the contraction. Phenylephrine-induced contraction was significantly less in the aortae of DIO mice than those of control mice; only GFX but not heparin attenuated the contraction, indicating a diminishing role of IP3 receptor in DIO mice. Western blotting showed the reduced expression and phosphorylation of IP3 receptor and the down-regulated expression of PKC, PKCβ, PKCδ, and PKCζ in DIO mouse aortae. Importantly, PKCδ was more likely to maintain phenylephrine-mediated contraction in DIO mouse aortae because that (1) PKCδ inhibitor rottlerin but not PKCα and PKCβ inhibitor Gö6976, PKCβ inhibitor hispidin, or PKCζ pseudosubstrate inhibitor attenuated the contraction; and (2) PKCδ phosphorylation was increased but phosphorylations of PKCα, PKCβ, and PKCζ were reduced in DIO mouse aortae. The present study thus provides additional insights into the cellular mechanisms responsible for vascular dysfunction in obesity.

Soluble guanylyl cyclase (sGC), phosphodiesterase type 5 (PDE5), and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) are all dimeric. The present study was to determine the role of their dimeric status in nitric oxide-induced vasodilatation. In isolated porcine coronary arteries, after 20 h incubation with serum-free medium, serum-containing medium, or phosphate-buffered saline solution, the protein levels of the dimers of sGC, PDE5, and PKG were diminished while the monomer levels remained unchanged, associated with reduced cGMP elevation in response to DETA NONOate and decreased PDE5 activity; the activity of PKG was not significantly altered. DETA NONOate caused a greater relaxation in arteries incubated for 20 vs. 2 h. The relaxant response was largely abolished by 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor. Zaprinast, a PDE5 inhibitor, had no effect on relaxation caused by DETA NONOate of arteries incubated for 20 h but augmented the response incubated for 2 h. A greater relaxation to 8-bromo-guanosine 3'5'-cyclic monophosphate occurred in arteries incubated for 20 than for 2 h. The protein level of the dimers but not monomers of PDE5 was reduced by dithiothreitol and unaffected by hydrogen peroxide, accompanied with decreased PDE5 activity and reduced response to DETA NONOate. These results demonstrate that the dimeric but not monomeric status of sGC and PDE5 of coronary arteries are closely related to their activities. The preserved vasodilator response after 20 h incubation may result in part from a synchronous reduction of the dimer levels of sGC and PDE5 as well as an augmented response to cGMP.

The study of base station (BS) distribution model is critical in intensive deployment scenario of modern wireless communication especially in crowded metropolis. In this paper, the distribution pattern of BS location and neighbor distances were analyzed based on massive real data of BS locations in representative metropolis of France. Using Delaunay triangulation and fine classification, the BS distribution is verified to be non-Poisson with repulsive interaction between BS. And Strauss-Hardcore point process is the closest model to describe actual BS distribution. The studied stochastic characteristics can provide empirical suggestions for wireless network planning and optimization.

Purpose: Activating Q209L/P mutations in GNAQ or GNA11 (GNAQ/11) are present in approximately 80% of uveal melanomas. Mutant GNAQ/11 are not currently therapeutically targetable. Inhibiting key down-stream effectors of GNAQ/11 represents a rational therapeutic approach for uveal melanomas that harbor these mutations. The mitogen-activated protein/extracellular signal–regulated kinase/mitogen-activated protein kinase (MEK/MAPK) and PI3K/AKT pathways are activated in uveal melanoma. In this study, we test the effect of the clinically relevant small molecule inhibitors GSK1120212 (MEK inhibitor) and GSK2126458 (pan class I PI3K inhibitor) on uveal melanoma cells with different GNAQ/11 mutation backgrounds. Experimental Design: We use the largest set of genetically annotated uveal melanoma cell lines to date to carry out in vitro cellular signaling, cell-cycle regulation, growth, and apoptosis analyses. RNA interference and small molecule MEK and/or PI3K inhibitor treatment were used to determine the dependency of uveal melanoma cells with different GNAQ/11 mutation backgrounds on MEK/MAPK and/or PI3K/AKT signaling. Proteomic network analysis was done to unveil signaling alterations in response to MEK and/or PI3K small molecule inhibition. Results: GNAQ/11 mutation status was not a determinant of whether cells would undergo cell-cycle arrest or growth inhibition to MEK and/or phosphoinositide 3-kinase (PI3K) inhibition. A reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK + PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant–dependent manner. Conclusions: MEK + PI3K inhibition may be an effective combination therapy in uveal melanoma, given the inherent reciprocal activation of these pathways within these cells. Clin Cancer Res; 18(16); 4345–55. ©2012 AACR.

The recent identification of frequent activating mutations in GNAQ or GNA11 in uveal melanoma provides an opportunity to better understand the pathogenesis of this melanoma subtype and to develop rational therapeutics to target the cellular effects mediated by these mutations. Cell lines from uveal melanoma tumors are an essential tool for these types of analyses. We report the mutation status of relevant melanoma genes, expression levels of proteins of interest, and DNA fingerprinting of a panel of uveal melanoma cell lines used in the research community.

The present study was to determine the role of the type I isoform of cGMP-dependent protein kinase (PKG I) and its downstream effector myosin phosphatase target subunit 1 (MYPT1) in the responses of different sized coronary arteries to nitrovasodilators. Relaxations of isolated porcine coronary arteries were determined by isometric tension recording technique. Protein levels of PKG I and its effectors were analyzed by Western blotting. The activities of PKG I and MYPT1 were studied by analyzing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and MYPT1, respectively. Nitroglycerin, DETA NONOate, and 8-Br-cGMP caused greater relaxations in large than in small coronary arteries. Relaxations were attenuated to a greater extent by Rp-8-Br-PET-cGMPS (a PKG inhibitor) in large vs. small arteries. The expressions of PKG I and MYPT1 in large arteries were more abundant than in small arteries. DETA NONOate stimulated phosphorylation of VASP at Ser239 and inhibited phosphorylation of MYPT1 at Thr853 to a greater extent in large than in small arteries. A suppressed phosphorylation of MYPT1 at Thr853 was caused by 8-Br-cGMP in large but not small arteries, which was inhibited by Rp-8-Br-PET-cGMPS. These results suggest that the greater responsiveness of large coronary arteries to nitrovasodilators result in part from greater activities of PKG I and MYPT1. Dysfunction in nitric oxide signaling is implicated in the vulnerability of large coronary arteries to certain disorders such as atherosclerosis and spasm. Augmentation of PKG I-MYPT1 signaling may be of therapeutic benefit for combating these events.

Glucagon-like peptide-1 (GLP-1) exerts its actions via activating GLP-1 receptor (GLP-1R). Our previous study showed a reduced GLP-1R expression in spontaneously hypertensive rat (SHR) renal arteries. The present study investigated the mechanisms underlying GLP-1R downregulation in hypertension.Intrarenal arteries of normotensive Wistar-Kyoto rat (WKY) and SHR were suspended in the myograph for force measurement. GLP-1R expression was evaluated by both immunofluorescence and western blotting. Protein kinase Cα (PKCα), PKCβ, PKCδ, and total PKC levels were assayed by western blotting.Immunofluorescence revealed reduced GLP-1R level in SHR renal arteries compared with WKY renal arteries. GLP-1R agonist exendin-4 induced concentration-dependent relaxations in WKY arteries, which mainly depended on the presence of endothelium. GLP-1R antagonist exendin 9-39 inhibited this relaxation in WKY arteries, whereas the relaxations were significantly less in SHR arteries. Ex-vivo treatment with PKC inhibitor GFX, PKCα and PKCβ inhibitor Gö6976, and PKCβ inhibitor hispidin but not PKCδ inhibitor rottlerin improved the impaired relaxations and restored the diminished GLP-1R expression in SHR arteries. Furthermore, PKCβ level was greater in SHR than WKY arteries, with no difference in PKCα, PKCδ, or total PKC expressions between two rat strains. Treatment with PKC-activating agent phorbol-12-myristate-13-acetate attenuated exendin-4-induced relaxations and reduced GLP-1R expression in WKY arteries, which were reversed by GFX, Gö6976, or hispidin. More relevantly, immunofluorescence of human renal arteries also showed a reduced GLP-1R level in hypertensive patients.The present results provide novel evidence that the reduced GLP-1R expression in SHR renal arteries is most likely mediated through PKCβ upregulation; the latter probably contributes to the impaired GLP-1R-mediated vasorelaxations in hypertension.

Activation of endoplasmic reticulum (ER) stress in endothelial cells leads to increased oxidative stress and often results in cell death, which has been implicated in hypertension. The present study investigated the effects of berberine, a botanical alkaloid purified from Coptidis rhizoma, on ER stress in spontaneously hypertensive rats (SHRs) and the underling mechanism. Isolated carotid arteries from normotensive WKYs and SHRs were suspended in myograph for isometric force measurement. Protein phosphorylations and expressions were determined by Western blotting. Reactive oxygen species (ROS) level was measured by DHE staining. SHR carotid arteries exhibited exaggerated acetylcholine-triggered endothelium-dependent contractions (EDCs) and elevated ROS accumulation compared with WKY arteries. Moreover, Western blot analysis revealed the reduced AMPK phosphorylation, increased eIF2α phosphorylation, and elevated levels of ATF3, ATF6, XBP1 and COX-2 in SHR carotid arteries while these pathological alterations were reversed by 12 h-incubation with berberine. Furthermore, AMPK inhibitor compound C or dominant negative AMPK adenovirus inhibited the effects of berberine on above-mentioned marker proteins and EDCs. More importantly, ROS scavengers, tempol and tiron plus DETCA, or ER stress inhibitors, 4-PBA and TUCDA normalized the elevated levels of ROS and COX-2 expression, and attenuated EDCs in SHR arteries. Taken together, the present results suggest that berberine reduces EDCs likely through activating AMPK, thus inhibiting ER stress and subsequently scavenging ROS leading to COX-2 down-regulation in SHR carotid arteries. The present study thus provides additional insights into the vascular beneficial effects of berberine in hypertension.

In this paper, the mobile deployment and subchannel number distribution are estimated for different values of Anisotropy Ratio (AR) and Mean Building Block Area (MBBA) pairs. This work proposes to model city maps with a particular family of random tessellations: Crack STIT tessellation to generate realistic city maps with a reduced number of parameters: i.e., AR and MBBA. The model is used to compute the received power map and further identify the impact of terrain topology parameters on wireless propagation. Simulations results show that mean mobile number and mean sub-channel number (SCN) both decrease with increasing MBBA following a power law dependency. The variance of SCN distribution increases with increasing MBBA by a potential exponential law. However, AR does not show obvious and strong impact on mobile and SCN. For different value of MBBA, AR leads to different mobile number and sub-channel evolutions gradually. Furthermore, the evolution of SCN does not monotonically decrease with increasing MBBA at 95% cumulative density function (CDF) bound of SCN distribution. Unlike the mean value (50% CDF bound), the SCN at 95% bound increases back for very large MBBA. The results are particularly promising for providing a parametric relation between mobile deployment/SCN and terrain topology. This considerably simplifies the radio estimation and planning of wireless networks.

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

Rays in city canyon is the most common mode of radio propagation. Our research is also based on this kind of urban assumption. This paper studies the impact of spatial building distribution on the path-loss exponent. The results are obtained by simulation. The simulation is based on a 3D ray tracing algorithm for the computation of the received power and on a stochastic geometry process for the generation of buildings. The evolution of the path-loss exponent against several terrain parameters is investigated. Parameters such as spatial density, the distribution of buildings and the distribution of antennas heights are considered. Some interesting results were found, and statistical fittings are proposed for the path-loss exponent from the results obtained by simulation.