It is now widely accepted that stem cells exist in various cancers, including lung cancer, which are referred to as cancer stem cells (CSCs). CSCs are defined in this context as the subset of tumor cells with the ability to form tumors in serial transplantation and cloning assays and form tumors at metastatic sites. Mouse models of lung cancer have shown that lung CSCs reside in niches that are essential for the maintenance of stemness, plasticity, enable antitumor immune evasion, and provide metastatic potential. Similar to normal lung stem cells, Notch, Wnt, and the Hedgehog signaling cascades have been recruited by the CSCs to regulate stemness and also provide therapy-driven resistance in lung cancer. Compounds targeting β-catenin and Sonic hedgehog (Shh) activity have shown promising anti-CSC activity in preclinical murine models of lung cancer. Understanding CSCs and their niches in lung cancer can answer fundamental questions pertaining to tumor maintenance and associated immune regulation and escape that appear important in the quest to develop novel lung cancer therapies and enhance sensitivity to currently approved chemo-, targeted-, and immune therapeutics.

Small cell lung cancer (SCLC) is an extremely aggressive neuroendocrine tumor, accounting for approximated 13% of all lung cancer cases. SCLC is characterized by rapid growth and early metastasis. Despite marked improvements in the number and efficacy of targeted, therapeutic options and overall survival rates in SCLC have remained nearly unchanged for almost three decades. The lack of significant progress can be attributed to our poor understanding of the biology of SCLC. Although immune checkpoint inhibitors were recently approved as front-line therapies for SCLC, we still need to better understand the mechanisms responsible for the selective vulnerability of some SCLCs to these inhibitors. Recent work utilizing sequencing data and single cell analyses identified four distinct subsets of SCLC, based on the expression levels of the transcription factors ASCL1, NEUROD1, POU2F3 and YAP1. Each subset was found to have its own distinct biology and therapeutic vulnerabilities. However, these subsets appear to be phenotypically unstable, representing snapshots in the gradual evolution of a tumor that exhibits significant plasticity. Tumor evolution, a product of this plasticity, results in the emergence of significant intratumoral heterogeneity which plays an important role in multiple aspects of SCLC development and progression, including cell survival and proliferation, metastasis and angiogenesis. The recent paradigm shifting discoveries in the biology of SCLC are now beginning to inform the design of new therapeutic strategies for the management of this intractable disease.

IntroductionRelapsed SCLC is characterized by therapeutic resistance and high mortality rate. Despite decades of research, mechanisms responsible for therapeutic resistance have remained elusive owing to limited tissues available for molecular studies. Thus, an unmet need remains for molecular characterization of relapsed SCLC to facilitate development of effective therapies.MethodsWe performed whole-exome and transcriptome sequencing of metastatic tumor samples procured from research autopsies of five patients with relapsed SCLC. We implemented bioinformatics tools to infer subclonal phylogeny and identify recurrent genomic alterations. We implemented immune cell signature and single-sample gene set enrichment analyses on tumor and normal transcriptome data from autopsy and additional primary and relapsed SCLC data sets. Furthermore, we evaluated T cell-inflamed gene expression profiles in neuroendocrine (ASCL1, NEUROD1) and non-neuroendocrine (YAP1, POU2F3) SCLC subtypes.ResultsExome sequencing revealed clonal heterogeneity (intertumor and intratumor) arising from branched evolution and identified resistance-associated truncal and subclonal alterations in relapsed SCLC. Transcriptome analyses further revealed a noninflamed phenotype in neuroendocrine SCLC subtypes (ASCL1, NEUROD1) associated with decreased expression of genes involved in adaptive antitumor immunity whereas non-neuroendocrine subtypes (YAP1, POU2F3) revealed a more inflamed phenotype.ConclusionsOur results reveal substantial tumor heterogeneity and complex clonal evolution in relapsed SCLC. Furthermore, we report that neuroendocrine SCLC subtypes are immunologically cold, thus explaining decreased responsiveness to immune checkpoint blockade. These results suggest that the mechanisms of innate and acquired therapeutic resistances are subtype-specific in SCLC and highlight the need for continued investigation to bolster therapy selection and development for this cancer.

Abstract STK11 (liver kinase B1, LKB1) is the fourth most frequently mutated gene in lung adenocarcinoma, with loss of function observed in up to 30% of all cases. Our previous work identified a 16-gene signature for LKB1 loss of function through mutational and nonmutational mechanisms. In this study, we applied this genetic signature to The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples and discovered a novel association between LKB1 loss and widespread DNA demethylation. LKB1-deficient tumors showed depletion of S-adenosyl-methionine (SAM-e), which is the primary substrate for DNMT1 activity. Lower methylation following LKB1 loss involved repetitive elements (RE) and altered RE transcription, as well as decreased sensitivity to azacytidine. Demethylated CpGs were enriched for FOXA family consensus binding sites, and nuclear expression, localization, and turnover of FOXA was dependent upon LKB1. Overall, these findings demonstrate that a large number of lung adenocarcinomas exhibit global hypomethylation driven by LKB1 loss, which has implications for both epigenetic therapy and immunotherapy in these cancers. Significance: Lung adenocarcinomas with LKB1 loss demonstrate global genomic hypomethylation associated with depletion of SAM-e, reduced expression of DNMT1, and increased transcription of repetitive elements.

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition.

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.

In the USA approximately 15% of the patients with lung adenocarcinoma have tumors associated with "driver" mutations in the EGFR gene that demonstrate major clinical responses to EGFR Tyrosine Kinase Inhibitors (EGFR TKIs). However, despite the fact that these mutations are always "truncal" (present in every tumor cell), and dramatic tumor shrinkage is seen initially in almost all patients, EGFR TKIs are never curative and tumors always recur. Some tumors that develop resistance appear to have pre-existing resistant sub clones, but the majority appear to acquire resistance by mutational target reactivation or bypass mechanisms. In order to develop acquired resistance, a subset of cells must survive at initial stage of therapy ("drug persister cells", or DPCs) which are known to serve as a reservoir for accumulation mutation rendering drug resistance. We have demonstrated that Notch3 mediated β-catenin activation enables drug persistence, an essential step for the development of drug resistance. We have conducted tumor protein expression analysis on a tissue micro array (TMA) containing 86 NSCLC tumors obtained before therapy. These TMAs were subjected to IHC analysis to detect the expression of Notch3 and β-catenin signaling which is known to regulate drug persistence. We have also performed cell viability and biochemical assays under various perturbations to study drug persistence mechanisms in EGFR mutant NSCLC cells. Previously, we have demonstrated that EGFR TKI treatment leads to drug persistence through Notch3 mediated β-catenin activation. Using pre-treatment NSCLC patient tissue micro array (TMA) we identified that there is frequent co-expression of Notch3 and β-catenin (total) proteins in 90% of EGFR mutant NSCLC. We also observed that there are a relatively low proportion (10%) of patients with active β-catenin in these pretreatment samples. This suggests that the EGFR mutant tumors upregulate Notch3 protein expression, but that β-catenin is predominantly transcriptionally inactive before EGFR TKI treatment. We have also identified that Notch3 is a novel transcriptional target of β-catenin which in turn promotes both β-catenin and EGFR stability. These findings suggest that the Notch3 and inactive β-catenin co-expression is characteristic of EGFR mutant tumors, and β-catenin activation at baseline is infrequent. EGFR TKI therapy activates β-catenin signaling in a Notch3 dependent manner, and here we show that both proteins are frequently highly expressed in these tumors. The concept of DPCs serving as a reservoir for accumulation of mutations that could cause drug resistance is novel. However, signaling pathways that are associated with the activation of drug persistence are not well characterized. For the first-time, our studies demonstrate that Notch3, β-catenin andEGFR regulate each other and EGFR TKI therapy mediated Notch3 activation leads to β-catenin activation which is essential for the maintenance of drug persister cells in a positive feedback loop. By understanding and targeting the Notch3 - β-catenin axis that control DPCs, these studies can develop therapeutics to prevent resistance to EGFR TKI therapy.

Lung cancer management has been transformed by the discovery and effective targeting of driver mutations such as epidermal growth factor receptor (EGFR). Tyrosine Kinase Inhibitors (TKIs) that block EGFR kinase activity such as erlotinib and osimertinib cause dramatic tumor shrinkage and prolonged responses in patients whose tumors have activating EGFR mutations. However, resistance universally develops. Drug persister cells with properties of cancer stem cell-like cells play a major role in drug resistance. In tumors without pre-existing resistant clones, some tumor clonogens survive, despite expressing the driver mutation, to acquire target reactivation or bypass resistance mechanisms. Identification of biomarkers that are mechanistically linked to EGFR TKI resistant cell populations would have clinical utility in early determination of disease recurrence in EGFR mutant NSCLC patients. Previously, we have reported that EGFR-TKI treatment activates Notch3, and here we have identified that Notch3 activates β-catenin transcriptional target, PAI1, which could play a role in drug persistence and could be used for early identification of disease recurrence. Our in vitro ELISA studies have identified an increase in secreted PAI1 levels after treatment with the EGFR TKIs erlotinib and osimertinib demonstrate that measuring PAI1 levels could serve as an indicator of EGFR TKI persistence. To further validate PAI1 as a surrogate biomarker for EGFR TKI treatment, we performed a retrospective study in NSCLC patients (n = 42) with EGFR mutant tumors. Using ELISA to detect PAI1 protein in pre- and post-treatment serum samples as a mechanism-based indicator for β-catenin activation, we observed a broad range in pre-treatment PAI1 levels, spanning from 0.8 ng/ml to 134 ng/ml, suggesting a variable baseline β-catenin activation. Doing survival analysis after dichotomizing patients into low and high baseline PAI1 level groups, we found patients with low basal PAI1 levels had a significantly shorter PFS with EGFR TKI treatment compared to patients with high basal levels. Comparing patients with a ≥ 2-fold induction to those with less than 2-fold, there was a similar trend for worsened survival in those who showed significant induction of PAI1. Overall, we demonstrate that PAI1 expression is a candidate marker for β-catenin activation and the development of drug persisters; thus, it is a potential surrogate marker for poor outcome in patients receiving EGFR TKI therapy and a potential biomarker for selecting patients for β-catenin-targeted therapy. Citation Format: Rajeswara Rao Arasada, Walter Wang, Shankar Suman, David Paul Carbone. PAI1, a β catenin transcriptional target, serves as a surrogate biomarker to predict EGFR TKI mediated drug persistence in EGFR mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4930.

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1–3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4–6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7–9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy. Loss of RB promotes both malignant progression and the development of metastatic disease; however, whereas reactivation of the RB pathway can revert metastatic tumour cell states to non-metastatic cell states, malignant cell proliferation is supported by MAPK–CDK2-dependent suppression of RB.

Tumor suppressor genes play critical roles orchestrating anti-cancer programs that are both context dependent and mechanistically diverse. Beyond canonical tumor suppressive programs that control cell division, cell death, and genome stability, unexpected tumor suppressor gene activities that regulate metabolism, immune surveillance, the epigenetic landscape, and others have recently emerged. This diversity underscores the important roles these genes play in maintaining cellular homeostasis to suppress cancer initiation and progression, but also highlights a tremendous challenge in discerning precise context-specific programs of tumor suppression controlled by a given tumor suppressor. Fortunately, the rapid sophistication of genetically engineered mouse models of cancer has begun to shed light on these context-dependent tumor suppressor activities. By using techniques that not only toggle “off” tumor suppressor genes in nascent tumors, but also facilitate the timely restoration of gene function “back-on again” in disease specific contexts, precise mechanisms of tumor suppression can be revealed in an unbiased manner. This review discusses the development and implementation of genetic systems designed to toggle tumor suppressor genes off and back-on again and their potential to uncover the tumor suppressor’s tale.

We have identified a non-canonical role of0 Notch3 in response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy, whereby Notch3 associates with β-catenin, resulting in increased catenin beta-1 (CTNNB1, best known as β-catenin) stability and increased survival of drug persister cells (DPCs). Furthermore, combined treatment of an EGFR TKI with a β-catenin inhibitor demonstrated improved therapeutic outcomes in xenograft models.

Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably though, Rb’s role in suppressing lung adenocarcinoma remains unclear, and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an Rb XTR allele that enables Cre-dependent inactivation of Rb in developing tumors, and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the Kras Lox-Stop-Lox-G12D/+ ;p53 flox/flox ( KP ) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although, in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest that a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types. This abstract is also being presented as Poster A27. Citation Format: Travis Yates, Caroline Kim-Kiselak, Walter Wang, David M. Feldser. Modeling Rb loss and pathway reactivation in lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR01.

Mutations in the Rb tumor suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma. Additionally, recent clinical successes with cyclin dependent kinase inhibitors have reinvigorated interest in reactivating the Retinoblastoma (Rb) pathway to treat lung adenocarcinoma and other tumor types. Remarkably though, Rb’s role in suppressing lung adenocarcinoma remains unclear, and whether Rb pathway reactivation would be efficacious in this disease remains unknown. To model Rb pathway reactivation as treatment strategy in lung adenocarcinoma and to shed light on its role in this disease, we established an Rb XTR allele that enables Cre-dependent inactivation of Rb in developing tumors, and allows Flp recombinase-inducible reactivation of Rb after tumors are established. In the Kras Lox-Stop-Lox-G12D/+ ;p53 flox/flox ( KP ) mouse model of lung adenocarcinoma, we show that Rb inactivation facilitates the bypass of two molecularly distinct barriers to tumor progression and dramatically accelerates malignant conversion and the development of metastatic disease. Although, in the presence of Rb, malignant conversion requires amplification of the Raf/Mek/Erk (MAPK) signaling pathway beyond that normally activated by the Kras oncogene, we find that this requirement is abrogated when Rb is inactivated. Mechanistically, we identified Cdk2 as an important effector downstream of amplified MAPK signaling and that this activity suppresses Rb’s ability to limit the adenoma-to-carcinoma transition. Importantly, inactivation of Cdk2 reduces cell proliferation in Rb wild-type cells and confers sensitivity to Cdk4/6 inhibition in both human and mouse lung adenocarcinoma cell lines were intrinsically resistant. Acquiring metastatic competency in Rb wild-type tumors is causally linked to epigenetic changes resulting in loss of lung lineage cell fate-determining transcription factors and concomitant derepression of factors normally restricted to embryonic cell types. However, inactivation of Rb uncouples the onset of metastatic competency from the loss of lung lineage factors, facilitates the early derepression of prometastatic factors, and significantly enhances metastatic proclivity. Finally, we demonstrate that reactivation of Rb in metastatic disease settings reprograms these tumors toward a less aggressive cell state and improves overall survival. Our study highlights an unappreciated role for Rb in regulating metastasis-promoting programs, and the potential of Rb restorative therapies to treat lung adenocarcinoma. Further, we suggest that a renewed investment in the development of specific Cdk2 inhibitors may be necessary for Rb pathway reactivation in certain cancer types. This abstract is also being presented as Poster A27. Citation Format: Travis Yates, Caroline Kim-Kiselak, Walter Wang, David M. Feldser. Modeling Rb loss and pathway reactivation in lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR01.

EGFR tyrosine kinase inhibitors cause dramatic responses in EGFR-mutant lung cancer, but resistance universally develops. The involvement of β-catenin in EGFR TKI resistance has been previously reported, however, the precise mechanism by which β-catenin activation contributes to EGFR TKI resistance is not clear. Here, we show that EGFR inhibition results in the activation of β-catenin signaling in a Notch3-dependent manner, which facilitates the survival of a subset of cells that we call "adaptive persisters". We previously reported that EGFR-TKI treatment rapidly activates Notch3, and here we describe the physical association of Notch3 with β-catenin, leading to increased stability and activation of β-catenin. We demonstrate that the combination of EGFR-TKI and a β-catenin inhibitor inhibits the development of these adaptive persisters, decreases tumor burden, improves recurrence free survival, and overall survival in xenograft models. These results supports combined EGFR-TKI and β-catenin inhibition in patients with EGFR mutant lung cancer.

Previous studies showed that soluble Guanylate Cyclase (sGC) activation with BAY 60 protected against postischemic inflammation when injected as a preconditioning agent in wild type (WT) H129 and heme‐oxygenase‐1 KO (HO‐1‐/‐) mice. This occurred when given both 10 min and 24 hours prior to ischemia (Wang et al. AJP Heart Circ Physiol 305:H521‐H532, 2013). We hypothesized that the same protective effect would occur when BAY 60 is administered during ischemia. When tested we observed reduced postischemic leukocyte rolling (LR) and leukocyte adhesion (LA) to intestinal venules in WT and HO‐1‐/‐ mice, as well as prevention of increased TNF‐α and circulating neutrophil levels that accompany ischemia/reperfusion (I/R). Based on these findings, we further hypothesized that BAY 60 when given during ischemia would prevent intestinal mucosal mitochondrial dysfunction induced by I/R. Mitochondrial integrity was evaluated by the Calcein AM fluorescence method (used to measure mitochondrial permeability), and the JC‐1 fluorescence ratio (used to measure mitochondrial membrane potential). Both mitochondrial permeability and membrane potential were disrupted following I/R, which indicates the formation of mitochondrial permeability transition pores (mPTP). However, both Calcein fluorescence and JC‐1 fluorescence ratio were maintained at control levels in cells from the mice that were treated with BAY 60. Our results indicate that sGC activation during ischemia protects against I/R inflammatory processes and preserves mucosal mitochondrial function by preventing mPTP formation Supported by: HL095486 & AA14945. Bayer Schering Pharma AG provided BAY 60 as a gift.

Our previous studies showed that activation of soluble Guanylate Cyclase (sGC) protects against both postischemic intestinal and cardiac injury in wild type, heme‐oxygenase‐1 ‐/‐ (KO), and eNOS KO mice. The aim of this study was to determine whether a similar protective effect of sGC activation by BAY 60‐2770 (BAY 60) would occur in postischemic brain. In this study, leukocyte rolling (LR) and adhesion (LA) were quantified in single pial venules of male wild‐type (H129) mice using intravital fluorescence microscopy after two and three hours of reperfusion following 20 min of ischemia produced by bilateral common carotid artery occlusion. Levels of tumor necrosis factor‐a (TNF‐a) were measured in brain samples obtained after three hour reperfusion. Preconditioning with BAY 60 either 10 min (acute) or 24 hr (delayed) prior to ischemia/reperfusion (I/R) significantly reduced postischemic LR but had no effect on LA at both two and three hour reperfusion, while the elevated TNF‐a levels induced by brain I/R were significantly attenuated in both the hippocampus and cerebrum. These findings indicate that both acute and 24h BAY 60 treatment prior to brain I/R limits postischemic inflammation, and may have therapeutic value for suppressing stroke‐mediated damage. Supported by grants from the NIH (AA‐014945 and HL‐095486). BAY 60‐2770 was a gift from Bayer Schering Pharma AG.

Previous studies showed that soluble Guanylate Cyclase (sGC) activation protects against postischemic reperfusion inflammation when used as a preconditioning compound in wild type (WT) H129, heme‐oxygenase‐1 KO (HO‐1 −/−) and eNOS −/−mice. We hypothesized that the same protective effect would occur when administered near the end of an ischemic episode. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intestine. Circulating neutrophil counts were done using a hemo‐cytometer while TNF (Tumor Necrosis Factor)‐α was measured by an enzyme‐linked immunosorbent assay. The intra venous administration (30μg/kg) of BAY 60–2770 (sGC activator) during the last 5 min of the 45 min ischemic episode caused the postischemic LR and LA to remain at sham levels in the three strains of mice. Increases in intestinal TNF‐α and circulating neutrophils were also prevented. These results indicate that treatment with a sGC activator before initiating reperfusion can convey protection from an inflammatory response in WT, in mice with an impaired antioxidant system, and in the absence of expressed eNOS. This may provide an important strategy to minimize the damage done by and during treatment of occlusive vascular disease. Supported by: P01‐HL 095486 & R01‐AA14945. BAY 60–2770 was a gift from Bayer Schering Pharma AG.

Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²⁺. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function.

Our previous studies showed that soluble Guanylate Cyclase (sGC) activation protects against postischemic intestinal injury when used as a preconditioning stimulus in wild type, heme‐oxygenase‐1 KO and eNOS KO (eNOS −/−)mice. The aim of this study was to determine whether a similar protective effect of sGC activation by Bay60–2770 (Bay 60) would occur in postischemic cardiac injury. Infarct size (% LV), assessed by 2,3,5‐triphenyltetrazolium chloride (TTC) staining, was significantly increased in eNOS −/− mice after 30 min left anterior descending coronary artery (LAD) ischemia and 24 hr reperfusion as compared with eNOS −/− sham mice (p < 0.001). Bay 60 treatment (300ug/kg, ip bolus, 24hr prior to LAD ischemia) significantly attenuated I/R‐induced increase in infarct size (p < 0.05) in eNOS−/− mice. Serum Troponin‐I (cTnI), a marker of myocardial injury, after 30 min LAD ischemia and at 4 hr reperfusion, was markedly elevated in eNOS −/− mice, compared with eNOS −/− sham mice (p < 0.01). Preconditioning with Bay 60 24hr prior to cardiac I/R did not prevent the increase in cTnI levels. These findings indicate that Bay 60 preconditioning 24hr prior to cardiac I/R limits the size of the evolving infarct area at 24 hr of perfusion independently of eNOS and factors leading to early release of cTnI. Supported by a grant from the NIH (AA‐014945). BAY 60–2770 was a gift from Bayer Schering Pharma AG.

It was observed that heme oxygenase‐1 KO mice (Hmox1(−/−)), do not exhibit protection from ischemia/reperfusion (IR) inflammation afforded by hydrogen sulfide or ethanol, preconditioning stimuli that limit postischemic inflammation via soluble guanylate cyclase (sGC)‐dependent mechanisms in wild‐type mice (WT). BAY 60‐2770 activates oxidized/heme‐free sGC, while BAY 41‐2272 stimulates reduced sGC (JPET 335:85‐91, 2010). We hypothesized that pretreatment with the BAY compounds would limit postischemic inflammation & reverse nitrate tolerance in Hmox1(−/− ). Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intestine. Drug responses were measured on superior mesenteric arterial (SMA) rings isolated after IR. Preconditioning with either Bay compound 10 min prior to IR reduced postischemic LR and LA in both WT and Hmox1(−/−), and was associated with increased SMA responses to acetylcholine and sodium nitroprusside. Preconditioning 24 h prior to IR attenuated IR‐induced LR and LA in WT. 24 h experiments on Hmox1(−/−) are in progress. It is concluded that the sGC active agents, BAY 60‐2770 and BAY 41‐2272, limit postischemic inflammation and may also reduce nitrate tolerance in Hmox1(−/−). Supported by HL59976, HL82816 & AA14945. BAY 60‐2770 was a gift from Bayer Schering Pharma AG.