Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and an increased risk of progression to acute myeloid leukemia (AML). Despite advances in supportive and targeted therapies, disease-modifying interventions remain limited. Iron overload is increasingly recognized as a key driver of disease progression, amplifying oxidative stress and inflammation while impairing hematopoietic function. However, the mechanisms by which sustained iron excess contributes to disease evolution remain poorly understood. Hepcidin, the master regulator of systemic iron homeostasis, is produced by hepatocytes in response to iron and inflammatory cues and is negatively regulated by transmembrane protease serine 6 (TMPRSS6). Targeting TMPRSS6 to increase endogenous hepcidin offers a promising strategy to restrict iron overload and its inflammatory consequences. Here, we investigated hepatocyte-targeted silencing of Tmprss6 using a GalNAc-conjugated siRNA (SLN124) in the NUP98-HOXD13 (NHD13) mouse model of MDS. MDS and wild-type mice received monthly subcutaneous SLN124 (3 mg/kg) or oral deferiprone (1.25 mg/mL). Iron burden in MDS mice strongly correlated with ASC-speck formation in CD45+ hematopoietic cells, consistent with inflammasome activation. Both SLN124 and deferiprone reduced tissue iron deposition and ASC-speck abundance, with SLN124 producing the most pronounced effect. Long-term SLN124 treatment delayed disease progression and significantly prolonged survival, with 30% of treated mice surviving beyond 450 days compared with complete mortality by Day 420 in controls and deferiprone-treated mice. These findings demonstrate that Tmprss6 inhibition via SLN124 suppresses iron-driven inflammation, and mitigated disease progression in MDS mice, establishing TMPRSS6 silencing as a promising disease-modifying therapeutic approach.

Background One hemophilia treatment concept focuses on rebalancing coagulation and anticoagulation to restore normal blood clotting. Targeting the coagulation regulator, protein S (PS), in hemophilia shows promise to increase the generation of thrombin, a critical enzyme in the clotting process. Objectives This study aimed to: (1) assess whether inhibiting PS increases thrombin generation (TG) in plasma from individuals with hemophilia A (HA) and B (HB); and (2) develop a hepatocyte-targeted PS small interfering RNA (siRNA) therapy using N-acetylgalactosamine conjugation to restore hemostasis in hemophilia without increasing thromboembolic risks. Methods We assessed TG in plasma from patients with HA and HB. To target the liver specifically, we developed a PS-siRNA conjugated with N-acetylgalactosamine. This approach ensures that PS levels remain adequate in other cells, thereby minimizing the risk of thrombosis. Additionally, we evaluated the therapeutic potential of PS-siRNA in preclinical models. Results Inhibiting PS with a polyclonal antibody in plasma resulted in a 3- to 5-fold increase in TG in HA and a 4- to 9-fold increase in HB plasma, with a 70% reduction in plasma PS. In preclinical models, subcutaneous PS-siRNA therapy in HA mice and nonhuman primates successfully lowered PS levels and improved clot formation. It also prevented bleeding in both saphenous vein puncture and knee injury models in HA mice. Notably, it enhanced clotting without triggering widespread clot formation. Conclusion Reducing PS levels enhances TG in hemophilia models, and PS-siRNA therapy shows promise in improving hemostasis. This approach warrants further clinical investigation as a potential treatment for hemophilia.

Supplementary Table 1 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

This study identified advanced pharmacy practice experience (APPE) activities students wish to pursue via a qualitative analysis of goal setting. It also aimed to understand student goal setting alignment with core Entrustable Professional Activities (EPAs).SMART (specific, measurable, achievable, realistic/relevant, time-bound) goal data were reviewed, analyzed, and coded to develop an inductive thematic analysis. Overall frequency of SMART goal coding themes was analyzed using descriptive statistics, along with frequency of themes based on rotation type. Relative percentages of each rotation type were compared. A crosswalk between themes and EPA was created post-coding.Several themes among student SMART APPE goals from one complete academic year were identified (n = 79). A total of 1690 SMART goals were categorized into 22 themes. The top five most frequently utilized themes represent 60.6% of all goals, and included "Assess, Select, Recommend Therapy" (19%); "Patient Communication" (15.4%); "Foundational Knowledge" (12.4%); "Interprofessional Collaboration" (7.9%); and "Workflow, Roles and Responsibilities" (6%). The core EPA Patient Care Provider Domain was most common and accounted for 46.4% of SMART goals. The remainder included Information Master (18.8%); Practice Manager (16.1%); Interprofessional Education Team Member (7.9%); Population Health Promoter (6.6%); and Self-Developer (3.6%).Our analysis revealed that students set goals in alignment with the profession's primary focus of providing direct patient care however some EPAs were infrequently citied by students in their goal setting process which may mean there is less focus on these important skills.

Introduction Replacement factor therapy for hemophilia A (HA) and B (HB), inherited disorders caused by factor VIII (FVIII) or factor IX deficiency, has significant limitations: First, factor prophylaxis is administered intravenously (IV) more than once a week. Second, patients may still experience bleeding and, third, they may develop antibodies against these therapeutic agents. A bispecific antibody, Emicizumab, a functional analog of FVIIIa, has recently been approved in patients with HA, but additional treatment options for hemophilia are still needed. We reported that genetic ablation of anticoagulant protein S (PS) improves hemostasis in HA and HB mice and constitutes a novel therapeutic target (Blood 2018, 131:1360-1371). We also showed that partial reduction of PS by small interfering RNA conjugated to an N-acetylgalactosamine cluster (GalNAc-siRNA), exclusively targeting hepatocyte-expressed PS, protects mice with HA from acute hemarthrosis. (Blood 2020, 136 (Suppl 1): 20-21). Here, we studied hemostatic effects of partial PS reduction in human ex vivo HA models and characterized a novel GalNAc-siRNA drug candidate, SLN140, in a non-human primate (NHP) model of HA. Methods & Results Hemophilia severity correlates with the amount of thrombin generation (TG) in plasma. We therefore assessed the impact of PS lowering on TG in human platelet-free plasma (PFP) with low FVIII activity. Normal and PS-depleted PFP were mixed to obtain PFP samples with free PS levels from 0 to 94%. Addition of anti-FVIII antibody resulted in PFP with FVIII between 0 and 7%. TG assays were run with low tissue factor as a trigger. Reduction of free PS to 56% doubled peak TG in PFP containing 0-7% FVIII. Further PS reduction to 37% restored peak TG in PFP containing 0-7% FVIII, indicating that partial PS depletion is sufficient to normalize TG in HA. Since PS depletion (Stago) may also reduce tissue factor pathway inhibitor level, the next experiments were performed in normal PFP treated with anti-FVIII and anti-PS antibodies to achieve 0% residual FVIII activity and to lower free PS. Anti-FVIII antibodies lowered peak TG (10±0.1 nM vs 78±2 nM in untreated PFP). Concomitant addition of anti-PS antibodies to achieve 0% or 55% residual PS had similar impact of increasing TG peak (21±0.1 and 27±13 nM, respectively), endorsing that partial depletion of PS increases TG in PFP containing 0% FVIII (Fig 1A). These data were confirmed using severe HA patient derived-PFP. Addition of anti-PS antibodies resulted in an increase of peak TG (to 20±5 nM and to 20±8 nM at 0% and 55% free PS respectively, compared with 8±1 nM in untreated HA PFP), indicating that 45% PS reduction increased peak TG in severe HA PFP (Fig 1B). Finally, we assessed the effect of partial PS reduction by SLN140 in a NHP model of acquired HA (AHA). TG assays were run in the presence of 3.5nM soluble thrombomodulin to enable protein C activation and full assessment of all PS anticoagulant functions. AHA was induced by slow bolus IV injection of anti-FVIII antibody (20,000 BU/kg), which resulted in <0.5% FVIII activity and a reduction in peak TG compared to controls (16±3 nM vs 43±2 nM). NHPs then received either SLN140 or 0.9% NaCl subcutaneously (SC). A single dose of 3 or 10 mg/kg SLN140 reduced free PS level from day 14 to day 21 to about 45% and 25% of baseline, respectively (Fig 1C). At day 14, peak TG relative to baseline and endogenous thrombin potential were ~3-fold higher in the SLN140-treated groups than in the 0.9% NaCl treated group. On day 20, anti-FVIII antibody was injected into all NHPs and blood was collected after 4 hours. In this AHA model, SLN140 restored TG to normal range (peak TG relative to baseline: 0.9% NaCl: 18±8%; 3 mg/kg SLN140: 124±55%; 10 mg/kg SLN140: 123±68%, Fig 1D). Treatment with SLN140 was clinically well tolerated. Basic coagulation and safety parameters were not affected by PS reduction and were comparable in the control group and the 3 and 10 mg/kg SLN140 treatment groups (mean±SD): prothrombin time 14.3±1.0, 14.4±1.0, and 14.8±1.0 seconds; fibrinogen 2.6±0.5, 2.8±0.4, and 2.4±0.6 g/L; platelet count 417±65, 436±127, and 454±112 G/L; D-Dimer 134±41, 141±59, and 188±43 ng/mL, respectively. Conclusion PS reduction enhanced TG in human models of HA ex vivo and a single SC administration of SLN140 improved hemostasis in NHPs with severe HA. These results are encouraging for rebalancing hemostasis in hemophilia and support future clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BACKGROUND AND AIMS:The LPA gene encodes apolipoprotein (a), a key component of Lp(a), a potent risk factor for cardiovascular disease with no specific pharmacotherapy. Here we describe the pharmacological data for SLN360, a GalNAc-conjugated siRNA targeting LPA, designed to address this unmet medical need. METHODS:SLN360 was tested in vitro for LPA knockdown in primary hepatocytes. Healthy cynomolgus monkeys received single or multiple subcutaneous doses of the SLN360 sequence ranging from 0.1 to 9.0 mg/kg to determine the pharmacokinetic and pharmacodynamic effects. Liver mRNA and serum biomarker analyses were performed. RESULTS:In vitro, the SLN360 sequence potently reduces LPA mRNA in primary cynomolgus and human hepatocytes, while no effect was observed on the expression of APOB or PLG. In vivo, SLN360 exposure peaks 2 h after subcutaneous injection with near full elimination by 24 h. Specific LPA mRNA reduction (up to 91% 2 weeks after dosing) was observed with only the 3 mg/kg group showing appreciable return to baseline (40%). No consistent dose- or time-dependent effect on the expression of APOB, PLG or a panel of sensitive markers of liver lipid accumulation was observed. Potent (up to 95%) and long lasting (≥9 weeks) serum Lp(a) reduction was observed, peaking in all active groups at day 21. The minimally effective dose was determined to be 0.3 mg/kg with an ED50 of 0.6 mg/kg. CONCLUSIONS:SLN360 induces a sustained reduction in serum Lp(a) levels in cynomolgus monkeys following subcutaneous dosing. SLN360 has potential to address the unmet need of Lp(a) reduction in cardiovascular diseases.

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in β-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of β-thalassaemia and related disorders.

BACKGROUND & AIMS:Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). METHODS:Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. RESULTS:Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. CONCLUSIONS:CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. LAY SUMMARY:Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein.

Introduction & Aim: Hemophilia A (HA) is an X-linked disorder caused by an absence or a reduction of coagulation factor VIII. Patients with HA often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Hemarthrosis is caused by bleeding into joint spaces affecting the synovium, synovial blood vessels as well as cartilage and bone tissues. Current prophylactic treatments are not always effective and hemophilia patients can experience breakthrough bleeds. Recently, we demonstrated that inhibition of protein S (PS), a natural anticoagulant, controls coagulation and constitutes a potential therapeutic target in hemophilia (Blood 2018, 131:1360-1371). Here, we aim to translate our findings using small interfering RNA conjugated to an N-acetylgalactosamine (GalNAc) cluster to target Pros1 gene expression (GalNAc-PS siRNA) in vivo and exclusively in hepatocytes. siRNAs conjugated to a GalNAc cluster bind to asialoglycoprotein receptors expressed predominantly by hepatocytes thereby providing a potentially safe, specific and efficient delivery technology for therapeutic molecules. Methods & Results: Forty-two days after subcutaneous (s.c.) injection of GalNAc-PS siRNA (3mg/kg), wild-type (WT) mice were alive and did not display overt disseminated intravascular coagulation (DIC). In a second study in WT mice, DIC parameters assessed fourteen days after treatment with either 5mg/kg GalNAc-PS siRNA or with vehicle were also comparable between the two groups (platelet count: 578±284 vs 725±186 G/L, p>0.9, n=4-6; prothrombin time: 9.0±0.4 vs 8.9±0.3 seconds, p>0.9, n=4-6; fibrinogen: 1.5±0.5 vs 1.8±0.4 g/L, p>0.85, n=5-6; thrombin anti-thrombin complexes, TAT: 71±63 vs 115±34 μg/L, p>0.9, n=2-4) supporting that GalNAc-PS siRNA treatment can be safe. At the same time, mice treated with GalNAc-PS siRNA displayed lower plasma PS level compared to mice receiving the vehicle (52±12 vs 100±11 %, p<0.001, n=6). In the liver, PS mRNA levels were reduced by 69% compared to mice treated just with the vehicle (31±10 vs 100±24 %, p<0.0001, n=6). Importantly, in a murine model for hemophilia A (F8-/- mice) the intrinsically-activated test using ellagic acid (INTEM) assessed by rotative thromboelastometry (ROTEM®) was improved by treatment with GalNAc-PS siRNA (5mg/kg s.c) as compared to F8-/- mice treated with the vehicle (clotting time: 281±193 vs 802±330 seconds, p<0.01, n=6-11; clot formation time: 109±80 vs 657±466 seconds, p<0.05, n=6-11; alpha angle: 70±13 vs 35±24 mm, p<0.1, n=6-11). To assess if targeting PS using GalNAc-siRNA-PS protects mice from acute hemarthrosis (AH), we applied an AH model to F8-/- mice. Five days after injecting a single dose of 5mg/kg GalNAc-PS s.c., right knees were injured using a 30 gauge-needle and knee diameters were measured 72 hours later. Macroscopically, vehicle treated F8-/- mice developed extensive bleeding in injured knees as compared to GalNAc-siRNA-PS treated mice. Scores for intra-articular bleeding (2.4±0.9 vs 1.0±0.7, p=0.035, n=5-9) and synovial hyperplasia (2.4±0.9 vs 0.6±0.9, p=0.027, n=5-9) were higher in F8-/- mice treated by vehicle than in those treated by GalNAc-PS-siRNA. Moreover, knee joint swelling was reduced in GalNAc-siRNA-PS treated mice compared to those treated by vehicle (0.14±0.15 vs 0.78±0.50 mm, p=0.025, n=7-10). As expected, PS plasma levels were lower in GalNAc-PS siRNA treated mice compared to those which received vehicle (63±9 vs 101±19% of WT PS antigen level, p<0.0001, n=7-13) with no overt DIC (platelets count: 711±149 vs 681±189 G/L, p>0.9, n=7-12; prothrombin time: 8.5±0.3 vs 8.4±0.3 seconds, p>0.9, n=4-13; fibrinogen: 2.7±0.6 vs 3.0±0.8 g/L, p=0.73, n=7-12 and TAT: 33±42 vs 45±66 μg/L, p>0.9, n=6-10). Conclusion: These data provide the first evidence that using a GalNAc-siRNA conjugate to modulate Pros1 gene expression is well tolerated and has the ability to reduce plasma PS level and protect F8-/- mice from AH pointing to PS targeting using GalNAc-siRNA-PS as a new valuable therapeutic approach for hemophilia. Further analysis to understand if the inhibition of PS influences also the inflammatory processes causing the hemophilic arthropathy is ongoing. Disclosures Schaeper: Silence Therapeutics GmbH: Current Employment. Dames:Silence Therapeutics: Current Employment. Eisermann:Silence Therapeutics: Current Employment.

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNABid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicleHEPES) and siRNALuci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNABid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

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Introduction: SLN360 is a liver targeted GalNAc-conjugated siRNA developed for the treatment of cardiovascular disorders associated with elevated Lp(a). Lp(a) is a key independent, genetically dete...

Accumulation of excess iron in tissues causes organ damage and dysfunction and may lead to serious clinical consequences including liver cirrhosis, diabetes, growth retardation and heart failure. Iron overload is a major health threat in iron loading anemias, like beta-thalassemia, myelodysplastic syndrome and in hereditary hemochromatosis. In patients with beta-thalassemia major, iron overload develops due to frequent blood transfusions to control the severe anemia. In addition, iron overload also occurs in patients with beta thalassemia intermedia (non-transfusion dependent beta-thalassemia). In the later cases, iron overload develops through gastrointestinal iron hyperabsorption due to stressed and ineffective erythropoiesis. Importantly, expression of the peptide hormone hepcidin, which is the key modulator in iron homeostasis, is abnormally low and unable to block ferroportin-mediated intestinal iron absorption. In hereditary hemochromatosis, gene defects in the hepcidin-ferroportin axis controlling iron homeostasis, lead to hepatic iron overload. Therefore, in these indications, iron overload is caused by dysregulation or dysfunction of the hepcidin-ferroportin axis. Hepcidin is predominantly produced by the liver and is induced by activation of the BMP/SMAD signaling pathway. Furthermore, hepcidin is under the negative control of the transmembrane protease matriptase-2, encoded by the TMPRSS6 gene.

Abstract Purpose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node metastasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the pharmacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four different preclinical mouse models. Experimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes was tested in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous metastasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules (repeated low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. Primary tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistochemistry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to provide an insight into molecular changes on depletion of PKN3, in support of efficacy results. Results: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formation of spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well as in mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an increase in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change endothelial function, resulting in reduced colonization and micrometastasis formation. Conclusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation, which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy. Clin Cancer Res; 16(22); 5469–80. ©2010 AACR.

wnloaded pose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node tasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the acologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four nt preclinical mouse models. erimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes was in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous tasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules ted low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. ry tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistostry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to e an insight into molecular changes on depletion of PKN3, in support of efficacy results. ults: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formaf spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an se in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change helial function, resulting in reduced colonization and micrometastasis formation. clusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation, Con which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy.