Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Research Article

Manuela Aleku,1 Petra Schulz,2 Oliver Keil,1 Ansgar Santel,1 Ute Schaeper,1 Britta Dieckhoff,1 Oliver Janke,1 Jens Endruschat,1 Birgit Durieux,1 Nadine Ro¨der,1 Kathrin Lo¨ffler,1 Christian Lange,1 Melanie Fechtner,1 Kristin Mo¨pert,1 Gerald Fisch,1 Sibylle Dames,1 Wolfgang Arnold,1 Karin Jochims,3 Klaus Giese,1 Bertram Wiedenmann,2 Arne Scholz,2 and Jo¨rg Kaufmann1

1Silence Therapeutics AG; 2Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charite´-Universita¨tsmedizin, Berlin, Germany; and 3IASON Consulting, Niederzier, Germany

Abstract of chemically synthesized siRNA for therapeutic purposes (for a We have previously described a small interfering RNA (siRNA) technology review, see refs. 2, 4, 6–8). With respect to cancer, a delivery system (AtuPLEX) for RNA interference (RNAi) in the variety of nonviral nanoparticles (50–200 nm) have been recently vasculature of mice. Here we report preclinical data for reported to be suitable for RNAi-mediated tumor growth inhibition Atu027, a siRNA-lipoplex directed against protein kinase N3 in experimental mouse models (summarized in ref. 6). In most (PKN3), currently under development for the treatment of cases, various siRNA carriers such as cyclodextrin (transferrin advanced solid cancer. In vitro studies revealed that Atu027- receptor targeted; ref. 9), PEI (RGD-targeted PEG-PEI; ref. 10), mediated inhibition of PKN3 function in primary endothelial DOTAP (self-assembled LPD-nanoparticle; nanosized immunolipo- cells impaired tube formation on extracellular matrix and some; refs. 11, 12), AtuFECT (AtuPLEX; refs. 5, 13), or other cationic cell migration, but is not essential for proliferation. Systemic lipids (14–17) have been developed for efficient complexation and administration of Atu027 by repeated bolus injections or delivery of siRNAs. The addition of targeting ligands (transferrin, infusions in mice, rats, and nonhuman primates results in anisamide, RGD-peptide, poly-arginine, anti-TfR single-chain anti- specific, RNAi-mediated silencing of PKN3 expression. We body fragment/TfRscFv, DNA) and additional lipid moieties show the efficacy of Atu027 in orthotopic mouse models for (PEG-lipids, cholesterol, endosomolytic helper lipids, or peptides) prostate and pancreatic cancers with significant inhibition or the overall morphology of the generated nanoparticles of tumor growth and lymph node metastasis formation. The (characterized by charge and particle size) supposedly confer tumor vasculature of Atu027-treated animals showed a specific targeting specificity to either cancer cells and/or tumor vascula- reduction in lymph vessel density but no significant changes in ture. Likewise, siRNAs targeting the expression of oncogenes microvascular density. [Cancer Res 2008;68(23):9788–98] (EphA2, EWS-Fli-1, Raf-kinase, EGFR/Her2) or angiogenesis-promoting genes (VEGFR2 and CD31) were chosen for RNAi-mediated Introduction therapeutic intervention in oncology. It remains debatable by which mechanism siRNA-containing RNA interference (RNAi) can be used as novel therapeutic nanoparticles (>60 nm) can escape the circulation and cross the modality through specific silencing of therapeutically relevant endothelial barrier to efficiently penetrate the tumor tissue. genes in vivo (1–3). In particular, chemically synthesized, small However, in contrast to other organ parenchyma, the vascular interfering RNAs (siRNA) are currently used as a new class of bed (endothelial cells) is directly accessible for systemically therapeutic molecules, allowing the controlled down-regulation of administered particles and therefore predestinated to endocytosis pathologically relevant gene expression as for oncogenes in cancer of siRNA-loaded particles, offering the reasonable approach for (4). However, in analogy to classic antisense molecules the in vivo RNAi-based therapeutic modulation of the vasculature. Hence, an application of these molecules is not straightforward. The overall antiangiogenic approach by means of ‘‘formulated siRNA-inhibi- negative charge of siRNA molecules (up to 40 negative charges) and tor’’ targeting the tumor vascular compartment might bear the relatively high molecular weight (12,000–14,000 Da) prevent valuable strategic and therapeutic advantages. these from functional uptake of these novel therapeutic molecules We recently described a novel liposomal siRNA formulation in vivo. Besides the inefficient uptake and the degradation in based on cationic lipids (siRNA-lipoplex/AtuPLEX), containing endosomal compartments on the cellular level, unformulated neutral fusogenic and PEG-modified lipid components, for siRNAs are rapidly cleared by renal excretion from the blood improved pharmacokinetic properties, cellular uptake, and effi- stream when administered i.v. (5). cient siRNA release from the endosomes after endocytosis. Using To overcome these limitations, delivery technologies for this formulation, we have shown a selective inhibition of gene synthetic siRNAs are currently being developed, enabling the use expression in endothelial cells in vivo as shown by target-specific knockdown of CD31 [platelet endothelial cell adhesion molecule-1 (PECAM-1)] or TIE-2 in various mouse tissues (5, 18). Moreover, we Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). showed therapeutic efficacy in xenograft mouse tumor models Requests for reprints: Jo¨rg Kaufmann, Silence Therapeutics AG, Otto-Warburg after systemic administration of CD31 targeting siRNA-lipoplexes Haus, Robert-Ro¨ssle-Strasse 10, Berlin 13125, Germany. Phone: 49-30-9489-2833; (13). The observed efficacy was not related to toxic side effects Fax: 49-30-9489-2801; E-mail: [email protected]. I2008 American Association for Cancer Research. including unspecific stimulation of the innate immune system. doi:10.1158/0008-5472.CAN-08-2428 Repeated dosing of the AtuPLEX does not elevate systemic levels

Cancer Res 2008; 68: (23). December 1, 2008 9788 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Atu027 for RNAi-Based Cancer Therapy of cytokines such as IFN-a or interleukin (IL)-12 (5, 13). These Supplementary Table S1. All AtuPLEXes (siRNA-lipoplexes) were prepared results laid the foundation for the preclinical development of this as described previously (5). The physicochemical properties of Atu027 novel liposomal siRNA formulation for RNAi therapeutics in particles are summarized in Supplementary Fig. S1. In vitro oncology. transfection, immunoblotting, and functional assays. The siRNA-lipoplex containing 0.28 mg/mL siRNA (19 Amol/L) was further Here we discuss preclinical data on Atu027, a lipoplexed siRNA diluted to a final concentration of 1 to 20 nmol/L siRNA in 10% serum– molecule specifically targeting the expression of protein kinase N3 containing cell culture medium. Human HeLa, PC-3, and murine EOMA cell (PKN3). PKN3 has previously been identified as a downstream lines were obtained and cultivated according to the American Type Culture effector of the phosphoinositide-3-kinase (PI3K) signaling pathway Collection recommendation. Human umbilical vascular endothelial cells (19). Loss of PKN3 function in vascular and lymphatic endothelial (HUVEC) and human microvascular endothelial lung lymphatic cells cells by RNAi suggested PKN3 as an appropriate molecular target (HMVEC-LLy) were obtained from Lonza. for an antiangiogenesis therapeutic strategy. We show the thera- The following antibodies were used for immunoblotting: rabbit anti- peutic potential of Atu027 for tumor treatment in orthotopic mouse PTEN (Cell Signaling), rabbit anti-PKN3 (19), mouse anti–Hsp60 (BD tumor models along with PKN3 gene silencing in vivo. Furthermore, Laboratories), rabbit anti–Akt-1 (Cell Signaling), and mouse anti-tubulin we noticed changes in the tumor lymphatic vasculature on (Oncogene). For the Matrigel assay, HUVEC and HMVEC-LLy cells were Atu027 treatment, suggesting an anti-lymphangiogenesis mode of trypsinized 72 h posttransfection, counted, and seeded on Matrigel action for the inhibition of tumor growth and metastasis. basement membrane matrix (Becton Dickinson) with a seeding density of 100,000 per 24-well plate. At the same time point, cells were lysed for Western blotting to measure PKN3 protein expression. Photographs were Materials and Methods taken after 6 h to evaluate tube formation. In the transmigration (Boyden siRNA molecules and preparation of siRNA-lipoplexes. siRNA chamber) assay, HUVEC cells were trypsinized 72 h posttransfection, molecules (AtuRNAi, blunt-ended 23-mer, alternating 2¶-O-methyl modified) counted, and seeded onto Fluoroblok inserts in serum-free medium. Bottom were previously described (20) and were synthesized by BioSpring. To wells were filled with serum- and growth factor–containing medium as screen for PKN3 mRNA inhibition, eight different mouse and human chemoattractant. At the same time point, cells were lysed for Western specific siRNA molecules were designed by bioinformatics means with the blotting to show PKN3 knockdown. After 6 h, migrated cells were visualized human/mouse-specific PKN3(3) lead siRNA sequence, sense strand 1552- by staining with SYTOX Green (Molecular Probes), and photographs were agacuugaggacuuccuggacaa-1574 (NM_013355.3); for the rat studies, a rat taken to determine cell number. HUVEC cells were seeded for the scratch specific sequence was used, sense strand 5¶-aagcuugaggaauuccuugacaa-3¶ assay onto 24-well plates at 200,000 per well 48 h after transfection with (XM_216019); the Luciferase specific sequence, sense strand 5¶-aucac- siRNA-lipoplexes. The next day, confluent monolayers were scratched and guacgcggaauacuucga-3¶, was used as control. Complete sequence homology photographs were taken at 0, 3, and 6 h after scratching. In parallel, whole- between the PKN3(3) siRNA sequence and the corresponding cynomolgus cell lysates were generated for subsequent immunoblotting. PKN3 mRNA was confirmed by cloning and sequencing of the RNAi in vivo and tumor xenograft experiments in rodent species. corresponding cynomolgus cDNA derived from two different tissues (data Eight-week-old male NMRI-nu/nu mice (Harlan) and female nonobese not shown). A list of the siRNA sequences used in this study is shown in diabetic severe combined immunodeficient mice (Charles River) were used

Figure 1. Identification of PKN3-specific siRNAs and loss-of-function analysis in primary endothelial and lymph-endothelial cells. A, knockdown analysis for PKN3 protein expression in human (HeLa and HUVEC) and murine EOMA cell lines after transfection with eight different PKN3 targeting siRNA sequences [siRNAPKN3(1) to siRNAPKN3(8); ut, untreated] at indicated concentrations. After 48-h incubation, PKN3 inhibition was assessed by immunoblot analysis on whole-cell lysates; detection of Akt-1 served as control for equal protein loading. B, concentration-dependent inhibition of PKN3 protein expression with siRNAPKN3(3)-lipoplex (Atu027) in three human cell culture systems. Detection of PTEN protein served as control for equal protein loading. C and D, inhibition of tube formation on Matrigel with HUVEC (C) or HMVEC-LLy (D) cells treated with active [siRNAPKN3(3, 5)] and inactive [siRNAPKN3(1, 4)] siRNAPKN3-lipoplexes. Untreated (ut) and cells transfected with a Luciferase-specific siRNA (siRNALuc ) served as negative controls, indicating the formation of network like structures. Corresponding Western blots are shown below. PTEN served as protein loading control.

www.aacrjournals.org 9789 Cancer Res 2008; 68: (23). December 1, 2008

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Cancer Research in this study. For knockdown experiments, siRNA-lipoplex was administered amended on May 25, 1998, and published in the Federal Law Gazette part I, i.v. by low-pressure, low-volume tail vein injection at 300 AL/30 g mouse of No. 30, dated May 29, 1998. Male and female Cynomolgus monkeys received a lipoplex solution containing 0.28 mg/mL siRNA and 2.17 mg/mL lipid eight times either 270 mmol/L sucrose solution or Atu027 diluted in (equivalent to a dose of 2.8 mg/kg siRNA and 21.7 mg/kg lipid) for high dose. 270 mmol/L sucrose every 4th day by 4-h i.v. infusions. The administration For dose titration, siRNA-lipoplex was diluted in 270 mmol/L sucrose to volume was 10 mL/kg body weight/h. Five different Atu027 doses were keep the administration volume of 300 AL/30 g mouse constant. administered: 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg (dose levels refer to the siRNA). For infusion studies, mice and rats catheterized via jugular vein (Charles Animals were sacrificed 24 h after last infusion on day 30, and lung tissue River) received one daily 4-h i.v. infusion with lipoplexed siRNA solution samples were analyzed (n z 2 per group) by quantitative reverse containing 0.14 mg/mL siRNA and 10.9 mg/mL lipid (equivalent to a dose of transcription-PCR (RT-PCR) (TaqMan) and Quantigene 2.0 assay or by 2.8 mg/kg siRNA and 21.7 mg/kg lipid) of 20 mL/kg body weight for the high immunoblot for PKN3 mRNA and protein expression. dose on 4 consecutive days. For dose titration, siRNA-lipoplex solutions were qRT-PCR (TaqMan). For mRNA quantification, tissues were dissected diluted in 270 mmol/L sucrose to keep the administration volume constant. and instantly snap-frozen in liquid nitrogen. RNA isolation and TaqMan The orthotopic prostate tumor model was previously described (19, 21). RT-PCR have been described previously (5). The sequence of the primers in In the orthotopic pancreas tumor model, 1.0 Â 106 DanG cells/10 ALPBS TaqMan can be obtained on request. were injected into the pancreas parenchyma under total body anesthesia. Branched-chain DNA assay for measurement of RNA. Alternatively to All animal experiments were done according to approved protocols and in TaqMan, PKN3 mRNA levels in the monkey lung tissues were determined by compliance with the guidelines of the Landesamt fu¨r Gesundheit und means of the branched-chain DNA assay according to the manufacturer’s Soziales Berlin, Germany (no. G0077/05). protocol (QuantiGene Reagent System, Panomics). In vivo RNAi in nonhuman primates. The monkey experiments were Histologic analysis and determination of microvessel density and done at a certified German contract research organization. The study was lymphatic vessel density. Mice were sacrificed and tissues instantly conducted in compliance with Good Laboratory Practice regulations fixed in 4.5% buffered formalin for 16 h and processed for paraffin according to the ‘‘Chemikaliengesetz,’’ current edition, in Germany and embedding (PC-3 model) or mounted with optimum cutting temperature according to the ‘‘OECD Principles of Good Laboratory Practice’’ Document medium and instantly snap-frozen in liquid nitrogen (DanG model). Nos. 1 and 13 ENV/MC/CHEM(98)17, ENV/JM/MONO(2002)9. Animal care Paraffin sections (2 Am) or cryosections (10 Am) were cut and placed on was in compliance with the ‘‘Tierschutzgesetz’’ (German Federal Animal glass slides. Tissue sections were stained according to standard protocols Welfare Act) as promulgated on February 17, 1993, published in the with goat anti-CD31/PECAM-1 (Santa Cruz Biotechnology), rat anti-CD34 ‘‘Bundesgesetzblatt’’ (Federal Law Gazette) part I, No. 7 on February 26, 1993, (Cedarlane Labs), or rabbit anti–LYVE-1 (Angiobio for paraffin sections,

Figure 2. Loss of PKN3 has no effect on endothelial cell proliferation in vitro. A and B, growth curves for HUVEC (A) and HMVEC-LLy (B) cells after single transfection with lipoplexed siRNAPKN3(3) (Atu027) and siRNALuc at indicated concentration over a 5-d period (d2–d7). Cell numbers were determined by device-supported cell counting and respective knockdown of PKN3 monitored over time by Western blot. Cells were replated after 96 h in fresh medium during the course of the experiment. Hsp60 served as control for equal loading.

Cancer Res 2008; 68: (23). December 1, 2008 9790 www.aacrjournals.org

Reliatech for cryosections) to visualize blood or lymph endothelial cells in lipoplex treatment. PKN3 silencing did not lead to a significant the sections. The number of microvessels was determined by counting change in the proliferation status of endothelial HUVEC and CD31/CD34–positive vessels or LYVE-1–positive vessels in tumor sections HMVEC-LLy as well as cancerous HeLa and PC-3 cells (data not (22, 23). Sections were evaluated blinded by up to three investigators. shown) when compared with untreated or control siRNALuc- Four so-called ‘‘vascular hotspots’’ were identified at low magnification lipoplex–treated cells (Fig. 2A and B). Interestingly, PKN3 gene (Â25) and the number of vessels was counted at high magnification (Â200) with a Zeiss Axioplan light microscope. Vessel number as vascular silencing shown by Western blot was maintained up to 144 h in units was evaluated regardless of shape, branch points, and size lumens rapidly proliferating HUVEC and HMVEC-LLy after a single Atu027 (referring to ‘‘vessel per field’’). transfection (Fig. 2A and B, right). Taken together, Atu027 PKN3(3) siRNA plasma level profiling. Concentrations of the PKN3-siRNA [siRNA -lipoplex] target-specifically inhibits PKN3 gene ex- antisense and sense strand were measured independently in lithium- pression, and PKN3 seems to be dispensable for normal cell heparin plasma using a modified sandwich hybridization assay (24). The proliferation but rather plays a role in cell migration and/or method development for the siRNA quantitation assay will be published adhesion processes required for endothelial tube formation on 4 in detail elsewhere. extracellular matrix. To shed some light on the molecular F Statistical analysis. Data are expressed as means SEM. Statistical mechanism of PKN3 signaling in response to vascular endothelial significance of differences was determined by the Mann-Whitney U test. growth factor (VEGF)-A, we analyzed the phosphorylation status P < 0.05 was considered statistically significant. of diverse direct effectors of VEGF-R2 such as PI3K/Akt and phospholipase Cg/endothelial nitric oxide synthase after VEGF-A Results stimulation in nontransfected as well as siRNALuc- and siRNAPKN3(3)- Atu027-mediated PKN3 inhibition and in vitro loss-of- transfected HUVEC. No substantial differences in phophorylation function analysis in endothelial cells. PKN3 is a downstream levels were observed in PKN3-depleted and control HUVEC for these effector of the PI3K/PTEN signal transduction pathway (19). Here known effectors implicated in VEGF-A–mediated pathologic we set out to analyze the biological/physiologic consequences of angiogenic neovascularization (refs. 25, 26; Supplementary Fig. S3), PKN3 inhibition in cultured vascular endothelial cells and and no phosphorylation target could be identified thus far. lymphatic endothelial cells. First, we screened target-specific, Atu027-mediated RNAi in vivo. Systemic administration of blunt-ended, nuclease-stabilized (alternating 2¶-O-methyl modified) Atu027 resulted in suppression of PKN3 expression in vivo when siRNA molecules (AtuRNAi; ref. 20) targeting PKN3 expression in applied as bolus in mice or after (4 hours) infusion in mice, rats, respective cell lines. For both the in vitro and the subsequent and nonhuman primates. PKN3 mRNA expression was analyzed in in vivo applications, we used the previously described and different organs by quantitative RT-PCR (TaqMan) after repeated characterized siRNA-lipoplex formulations (AtuPLEX; for a detailed bolus administration of Atu027, control siRNA-lipoplex, and description, see ref. 5). Five of eight siRNA molecules (AtuRNAi; see sucrose solution. The PKN3 mRNA reduction was most robust in Materials and Methods) showed a marked decrease in PKN3 lung and liver tissues (Fig. 3A) and moderate in tumor tissues, but protein expression 48 h posttransfection (Fig. 1A) in HeLa cells and not apparent in heart and spleen. Importantly, we have previously two vascular endothelial cell lines, HUVEC along with EOMA shown that systemic administration of siRNA-lipoplexes (AtuPLEX) (mouse hemangioendothelioma endothelial cells). A titration of the led preferentially to RNAi in the mouse vasculature (5, 13). The lipoplexed siRNAPKN3(3) lead sequence, hereafter named Atu027, moderate PKN3 mRNA reduction observed for s.c. (DU-145) and revealed an IC50 of f5 nmol/L (concentration for siRNA) in HeLa, orthotopic (PC-3) prostate tumor xenografts might reflect the HUVEC, and HMVEC-LLy (Fig. 1B). PKN3 expression in nontargeted tumor cancer cells, which cannot HUVEC and HMVEC-LLy were treated with Atu027/siR- be reached with systemic AtuPLEX administration. In other words, NAPKN3(3)-lipoplex, another potent siRNAPKN3(5)-lipoplex, and two target mRNA reduction can be detected in whole highly negative siRNA-lipoplexes [siRNAPKN3(1) and siRNAPKN3(4)]or vascularized tissue lysates or even if target gene expression is control siRNALuc-lipoplex. The cells were seeded on extracellular either restricted to or up-regulated in endothelial cells. Therefore, it matrix 48 h posttransfection and analyzed (Fig. 1C and D). RNAi- is not surprising that Atu027-mediated inhibition of PKN3 mRNA mediated reduction of PKN3 expression in HUVEC and HMVEC- expression, which is not restricted to endothelial cells, can be LLy clearly impaired formation of the typical network-like shown most significantly in the highly vascularized lung. structures on Matrigel as observed for untreated or siRNALuc- Furthermore, we analyzed the gene silencing activity of Atu027 lipoplex–treated cells, suggesting PKN3 function in migration to be given at different doses in mice and rats by repeated daily i.v. required for the formation of endothelial cell tube–like structures. infusions (20 mL/kg body weight in 4 hours). In mice, four repeated To further analyze PKN3 participation in cell migration, we daily doses of 1.4 and 0.7 mg/kg Atu027 gave rise to a dose- examined HUVEC cells by transmigration assay (Boyden Chamber dependent significant mRNA reduction in the lung, whereas in the assay) and observed a marked decrease in transmigrated cells with liver 1.4 mg/kg showed the highest efficacy. Two low doses, reduced PKN3 expression (Supplementary Fig. S2A). This migration 0.35 and 0.18 mg/kg, did not show PKN3 mRNA reduction in any phenotype was confirmed in a scratch assay with HUVEC cells organ at all (Fig. 3B). We also monitored the siRNA levels in mouse treated with Atu027 (siRNAPKN3(3)-lipoplex; Supplementary plasma during the 4-h infusion twice (at 30 minutes and 3 h 50 Fig. S2B). Cells depleted of PKN3 (Supplementary Fig. S2B) showed minutes) and also 6 and 20 h postinfusion, using a modified impaired migration into scratch wounds as compared with controls. capture probe assay (see Material and Methods; Fig. 3B, bottom). To rule out any inhibition of cell proliferation as a direct Notably, peak plasma concentrations were f10 nmol/L during consequence of PKN3 loss-of-function phenotype in this assay, we infusion of the effective doses (1.4 and 0.7 mg/kg) and, therefore, PKN3(3) analyzed cell growth on plastic dishes after Atu027/siRNA - higher than the in vitro determined IC50 of f5 nmol/L. We performed the same study with rats, using a formulated homologous rat PKN3 targeting siRNA (four nucleotide changes in 4 C. Lange et al., in preparation. the 23-mer sequence). In rat, we observed PKN3 mRNA reduction www.aacrjournals.org 9791 Cancer Res 2008; 68: (23). December 1, 2008

Figure 3. PKN3 gene silencing in vivo after systemic administration of Atu027 [siRNAPKN3(3)-lipoplex] in mice and nonhuman primates. A, PKN3 mRNA levels determined by TaqMan real-time PCR after four bolus tail vein injections in different organs and s.c. and intraprostatic (iprost.) xenografts of prostate tumor tissue. Expression of PKN3 mRNA normalized to CD34 mRNA levels is shown as mean value for mice from individual treatment groups: 270 mmol/Lsucrose, Atu027 [siRNA PKN3(3)-lipoplex], unrelated control siRNACD31-lipoplex (control). *, P < 0.01, Mann-Whitney. B and C, PKN3 gene silencing after repeated 4-h i.v. infusions of three different doses Atu027 [siRNAPKN3(3)-lipoplex] as shown for liver and lung tissues in mice (B) and lung tissue in Cynomolgus nonhuman primates (C). The PKN3 mRNA knockdown was analyzed by real-time PCR and normalized to CD34 (B) and p110a (C) mRNA levels. Reduction of PKN3 protein level in lung extracts from treated Cynomolgus (C) is shown by immunoblot analysis for two individual animals per treatment group; p110a and actin served as loading controls. A branched-chain DNA (b-DNA) assay was carried out for showing dose- dependent PKN3 knockdown in Cynomolgus lung tissue (C, right, middle row). During infusion, the siRNA plasma concentrations for the indicated doses were determined for both strands (day 4 postinfusion in mice; B) or for the antisense strand on day 1 and day 29 postinfusion (monkey) and plotted as a function of time (B and C, bottom). Infusion periods are highlighted in gray.

in the lung and some modest target gene suppression in the liver at monkeys) as part of an initial 4-week subchronic toxicity study the highest dose (2.8 mg/kg), but not at lower doses as seen before for Atu027. Cynomolgus monkeys received, in the initial study, eight (Supplementary Fig. S4, compare with 3B). Therefore, the required repeated 4-h i.v. infusions of Atu027 at doses of 0.3, 1.0, and 3.0 mg effective dose seems to be higher in rats, even though the siRNA/kg every 4th day. Additionally, control animals were treated pharmacokinetic profile for the siRNA levels in rat plasma seemed in parallel with a 270 mmol/L sucrose carrier solution. Importantly, to be very similar to the one observed in mouse plasma in vivo gene silencing of PKN3 was observed when compared with (Supplementary Fig. S4, bottom). sucrose-treated animals for all three different doses on mRNA and Finally, we conducted a dose range finding study for assessing protein levels in monkey lung tissue samples harvested 24 h after RNAi in vivo efficacy in nonhuman primates (Cynomolgus the last administration on day 29 (Fig. 3C; PKN3 protein: top right;

Cancer Res 2008; 68: (23). December 1, 2008 9792 www.aacrjournals.org

Figure 3 Continued.

protein loading controls, p110a and actin; middle row left; Pkn3 with sucrose solution, Atu027, or control siRNALuc-lipoplex after mRNA normalized to ubiquitously expressed p110a mRNA). For tumor establishment with eight consecutive bolus injections every determination of the lowest RNAi-active dose, a second study in other day at a given dose (Fig. 4A). Notably, we did not observe Cynomolgus was carried out including two additional lower doses significant treatment-related changes in body weight in this at 0.03 and 0.1 mg siRNA/kg. Notably, dose-dependent silencing of experiment (Fig. 4A, top right). Tumor volumes and lymph node PKN3 expression was observed, revealing 0.3 mg/kg siRNA as the metastasis volumes and numbers were determined for one group minimal active dose in monkey (Fig. 3C, b-DNA). before treatment (pretreatment control group; day 35) to define Bioanalytic examination was done with whole blood from these tumor progression during the treatment period. Regarding monkey specimens for recording siRNA plasma concentrations at therapeutic efficacy, mean tumor and lymph node metastasis different time points during infusion on day 1 and day 29 to volumes further increased in the sucrose- and siRNALuc-treated determine possible changes in siRNA plasma clearance after control group, whereas Atu027/siRNAPKN3(3)-lipoplex treatment repeated dosing. The lipoplexed siRNA plasma profiles for the restrained further tumor growth and, more importantly, the infusion on day 1 and day 29 were similar for all five tested doses, formation of lymph node metastases (Fig. 4B). indicating that a repeated dosing is feasible (Fig. 3C, bottom). To show target-specific gene silencing in Atu027-treated mice, Interestingly, the siRNA plasma concentrations in monkeys were we determined the PKN3 mRNA level in lung tissue extracts 24 h significantly higher when compared with the plasma siRNA after last treatment by TaqMan-PCR. Down-regulation of PKN3 concentrations with similar doses administered to rodents mRNA in individual mice was detected in comparison with the (compare bottom graphs of Fig. 3B and C; Supplementary Fig. S3). control siRNALuc-lipoplex–treated mice (Fig. 4C). However, we were Tumor growth inhibition and reduction of lymph node not able to show a direct reduction of PKN3 target mRNA in tumor metastasis by systemic administration of Atu027. Having shown lysates, most probably due to the cell type heterogeneity within the RNAi in vivo activity of Atu027 in three different species, we tissue samples and/or the low expression levels of mouse PKN3 investigated the pharmacologic activity in tumor mouse models. mRNA in these low vascularized prostate tumors (data not shown). Metastases are responsible for the majority (f90%) of deaths The therapeutic efficacy and pharmacologic activity of Atu027 associated with solid tumors (27). Therefore, one major objective in were further proved in a study on PC-3 tumor inhibition using the treatment of solid cancer in humans is the prevention and a different treatment schedule (15 i.v. injections every 4th day inhibition of invasive tumor growth and metastasis. To test beginning treatment at day 7) at lower doses compared with the whether Atu027 has therapeutic potential in the prevention of 2.8 mg/kg siRNA dose used before (Fig. 5A). Repeated dosing every invasive tumor growth and metastasis, we used an orthotopic 4th day at 1.4 or 0.7 mg/kg, but not 0.3 mg/kg, siRNA resulted in prostate cancer (PC-3) model (19, 21). This model has the a significant inhibition of further tumor growth and lymph node advantage of analyzing both PC-3 prostate tumor growth and metastasis formation (Fig. 5B). At the same time, no signs of assessment of metastatic spread of cancer cells into adjacent toxicity or other adverse effects were observed over the entire lymph nodes. PC-3 cells were transplanted intraprostatically into treatment period, as indicated by the maintenance of stable nude mice, and animals (n = 8 per group) were treated as indicated bodyweight (Fig. 5A, right). www.aacrjournals.org 9793 Cancer Res 2008; 68: (23). December 1, 2008

Figure 4. Pharmacologic activity of Atu027 [siRNAPKN3(3)-lipoplex] in an orthotopic xenograft prostate cancer (PC-3) mouse model. A, experimental design, daily dose, and Atu027 treatment schedule. Right, body weight development during treatment. B, Atu027 inhibitory effects on the volume of established PC-3 xenograft tumors (left) and volume and number of lymph node (LN) metastases (middle and right) in comparison with control siRNALuc-lipoplex and vehicle (270 mmol/Lsucrose) and ‘‘pretreatment d35’’ control groups. Columns, mean volume from eight mice per treatment group; bars, SEM. *, P < 0.05, Mann-Whitney. C, in vivo PKN3 mRNA knockdown in lung tissue samples from individual Atu027-treated mice in comparison with levels observed in siRNALuc-lipoplex–treated animals. PKN3-specific mRNA reduction in total RNA was measured by real-time PCR and values were normalized to CD34 mRNA levels. *, P < 0.001, Mann-Whitney.

Furthermore, Atu027 was applied in additional mouse tumor tumors. Analysis of microvasculature density (MVD) or the models including experimental lung metastasis models (i.v. formation of lymph vessel structures [lymph vessel density injection of Lewis lung cancer cells and murine B-16V melanoma (LVD)] in tumor sections from animals treated with Atu027 was cells) and two different orthotopic pancreatic tumor models, carried out by staining with vessel specific markers such as anti- supporting the shown therapeutic efficacy (as summarized in CD34 or anti-CD31 (vasculature) and LYVE-1 (lymphatics). We used Supplementary Table S2). The broad therapeutic effect in these two different orthotopic tumor models, a PC-3 prostate cancer or a different models is consistent with the hypothesis that Atu027 DanG pancreatic cancer model, treated with the same daily dose mediates PKN3 gene silencing in the mouse vasculature and might of 2.8 mg/kg Atu027, but with different treatment schedules, interfere directly with angiogenesis, lymphangiogenesis, or metas- depending on the tumor model (Fig. 6A and C). A qualitative and tasis processes. quantitative determination of MVD using a CD34-specific (Fig. 6B) Atu027 reduces the lymphatic vessel density, but not or CD31-specific (Fig. 6D) antibody did not reveal a significant microvessel density, in two different mouse models. We set difference between Atu027-treated and sucrose-control animals out to determine whether PKN3 inhibition affects neoangiogenic (Fig. 6B and D, top) in the number of blood vessels. In contrast, processes during tumor progression and metastasis. Our previous analysis of LVD in corresponding sections of respective treatment studies with fluorescently labeled siRNA molecules indicated that groups revealed a significant reduction of lymphatic vessels in tumor vasculature is the preferred cellular target structure within tumor sections of the Atu027-treated animals (Fig. 6B and D,

Cancer Res 2008; 68: (23). December 1, 2008 9794 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Atu027 for RNAi-Based Cancer Therapy bottom). Although reduction of MVD was not detectable in these to be essential for cell migration and adhesion-dependent particular tumors, we currently cannot rule out a functional formation of network/tube-like structures on Matrigel in both impairment of tumor blood vessels. endothelial cell types. However, loss of PKN3 function did not significantly affect HUVEC or HMVEC-LLy cell proliferation. From these data, we reasoned that systemic RNAi-mediated PKN3 Discussion inhibition might prevent new vessel/endothelial cells from There is an unmet medical need for effective cancer therapies forming appropriate vessel structures without inhibiting the that block the progression of solid tumors and prevent the proliferation capacity, reducing the likelihood of target-related formation of metastasis. PKN3 has been validated as a toxic side effects caused by PKN3 inhibition in other cell types. therapeutic target in cancer cells downstream of PI3K signaling. The liposomal siRNA formulation used here (AtuPLEX) has À À For example, induced inhibition of PKN3 in PTEN / prostate been designed for systemic administration of siRNAs to treat cancer cells (PC-3) led to a reduced number of lymph node advanced solid cancers (5, 13). Consistent with our previous studies metastases in orthotopic prostate cancer models (19). In many on AtuPLEX-mediated siRNACD31 delivery, here we showed that human sporadic cancers, the PI3K pathway is chronically systemic Atu027 administration resulted in therapeutic efficacy in activated by loss of the tumor suppressor PTEN (28). In several tumor mouse models. The therapeutic effect is in particular endothelial cells, PI3K can be activated by a wide range of pronounced in the inhibition of lymph node metastasis (PC-3 vascular growth factors [e.g., VEGF, angiopoietins, basic fibroblast model) and invasive growth (see Supplementary Table S2; data not growth factor (bFGF), platelet-derived growth factor-B, ephrin-B2, shown). In parallel, we succeeded to show Atu027-mediated RNAi and transforming growth factor-h superfamily members] or in in vivo after systemic administration of siRNA by measuring target conjunction with shear stress leading to activation of neo- mRNA quantity in the surrogate lung tissue from three different vascularization (including tumor angiogenesis; ref. 29). Moreover, species. Because PKN3 is also highly expressed in nontargeted cells a recent publication suggested also a direct function of PI3K in of the primary tumor, reduction in PKN3 expression within the the control of endothelial cell migration during tumor angiogen- tumor endothelial cells cannot be easily detected without esis (30). We therefore rationalized that RNAi-mediated gene enrichment of this particular cellular entity. silencing of PKN3, as a downstream effector of PI3K, in the Tumor angiogenesis, the new generation or modulation of tumor vasculature of mice may result in inhibition of PI3K-dependent vessels, has been recognized as a hallmark step during carcino- tumoral hemangiogenesis and/or lymphangiogenesis. To support genesis, promoting tumor metastasis (31–33). In view of this, this hypothesis, we validated the PKN3 loss-of-function phenotype vascularized human tumors such as non–small-cell lung cancer in primary HUVEC (vascular endothelial) and HMVEC-LLy have been found to metastasize early in carcinogenesis to regional (lymph-endothelial) cell culture systems. PKN3 function seemed lymph nodes, correlating with the degree of lymphangiogenesis

Figure 5. Dose-dependent inhibition of tumor growth and lymph node metastasis after systemic Atu027 treatment. A, mice were treated every 4th day with sucrose or three different indicated daily doses of Atu027 [siRNAPKN3(3)-lipoplex], starting on day 7 after intraprostatic PC-3 cell transplantation. Body weight development was monitored during treatment (right). B, inhibition of PC-3 tumor growth (left) as well as volume and number of lymph node metastases after Atu027 treatment in comparison with vehicle (270 mmol/Lsucrose). Columns, mean of six to nine mice per group; bars, SEM. *, P < 0.05, Mann-Whitney. www.aacrjournals.org 9795 Cancer Res 2008; 68: (23). December 1, 2008

Figure 6. LVD, but not MVD, seems to be reduced in orthotopic prostate and pancreas cancer tumors as revealed by immunohistologic staining of respective tumor tissue sections from Atu027- (lipoplexed siRNAPKN3) and sucrose-treated mice. A and C, experimental design and treatment schedules for the PC-3 prostate cancer (A)or the DanG pancreatic cancer (C) model. In addition, mean tumor volumes in the prostate (A) or the pancreas (C) model at end of treatment (day 48 or day 22 after cell transplantation, respectively) are shown. B and D, MVD and LVD assessment for tumor sections from vehicle (sucrose)– or Atu027-treated groups by counting CD31/ CD34–positive blood vessels and LYVE-1–positive lymph vessels. The number of blood and lymph microvessels was quantified per field tumor section (right). Columns, mean of 12 to 14 samples per group; bars, SEM. *, P < 0.05, Mann-Whitney. Two representative pictures of stained tumor sections displaying CD31/CD34–positive or LYVE-1–positive vessels in respective tumor entities are shown.

(34, 35). Additionally, in humans, tumor-associated lymphangio- treatment, which does not ultimately indicate for a missing genesis correlates with lymph node metastasis and prognosis of contribution of the vasculature on tumor progression. The lack of several tumor entities (36, 37). Our data on Atu027 implicate a changes in MVD at the end of treatment might reflect that existing function in controlling lymphatic vessel growth, making an anti- mature blood vessels were not disrupted, but additional neo- lymphangiogenic cancer therapy conceivable. Lymphangiogesis is angiogenic processes became blocked during treatment leading to currently being considered as an attractive alternative strategy for smaller tumors. To clarify this issue, tumors needed to be harvested preventing metastasis (as shown for targeting NRP2; ref. 38). It is from control and Atu027-treated animals at early and late time suggestive that our observed reduction in LVD, owing to Atu027 points. Interestingly, in contrast to Atu027, systemic treatment of treatment, may be the main reason for the reduced metastatic mice with bevacizumab, a VEGF neutralizing monoclonal antibody spread of PC-3 cells into regional lymph nodes (Fig. 4B and 5B). (for summary, see ref. 39), clearly resulted in blocked tumor vessel At the same time, MVD seemed to be unchanged after Atu027 growth and, consequently, reduced MVD at the end of treatment in

Cancer Res 2008; 68: (23). December 1, 2008 9796 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Atu027 for RNAi-Based Cancer Therapy our xenograft models (data not shown). Notably, even induction Fig. S6, and data not shown). Neither naked nor lipoplexed PKN3 of excessive angiogenesis after Dll4 inhibition was suggested to siRNA or unrelated siRNA carrying the 2¶-O-methyl-modification block tumor growth, contrasting the classic antivascular/ (20) showed any significant increase in the levels of these particular antiangiogenesis concept (i.e., MVD reduction) for effective cytokines after single and repeated dosing, supporting our initial anticancer therapy (40). Finally, the vessel normalization rather results. The 2¶-O-methyl modified 23-mer siRNA most likely can than the disruption of tumor vasculature has been implied to abolish any immunostimulatory effects, as suggested by other provide antiangiogenic therapy benefits (41). Additional systemic published reports (45–47). Therefore, it seems unlikely that TLR effects of Atu027 treatment and PKN3 inhibition, such as direct activation contributes to our observed tumor efficacy, supporting obstruction of circulating and/or disseminated tumor cells as an RNAi dependent, target-specific mode of action of Atu027. well as endothelial progenitor cells, macrophages, or other bone In conclusion, this study supports the further development of marrow–derived cells, can be proposed as contributing to the Atu027 as a novel siRNA formulation, targeting PKN3 expression to onset of metastasis (42–44). However, we have no direct impede progression of multiple solid cancers, including pancreatic evidence of RNAi-mediated inhibition of PKN3 in these and prostate cancer. Currently, Atu027 is being evaluated in toxicity particular cell types thus far. Finally, it should be noted that studies in nonhuman primates for upcoming clinical trials. liposomal formulation of a different potent PKN3 siRNA (Supplementary Fig. S5A; siRNAPKN3(71)) had a similar antitumor effect, as shown in another prostate cancer xenograft (LNCaP; Disclosure of Potential Conflicts of Interest Supplementary Fig. S5), supporting the proposed therapeutic All authors except P. Schulz, K. Jochims, B. Wiedenmann, and A. Scholz are PKN3 loss-of-function effect. employees of and have stock options in Silence Therapeutics AG. O. Keil, A. Santel, and A sequence and target-independent TLR-mediated antiangio- J. Kaufmann are named inventors on patent applications directed to PKN3. O. Keil and J. Kaufmann are named inventors on patent applications directed to AtuPLEX. K. genesis effect after local injection (intravitreal) of naked siRNAs Jochims received commercial research support from Silence Therapeutics AG. B. has been proposed recently (45). However, we have no evidence for Wiedenmann, A. Scholz, and K. Jochims were paid consultants during the relevant TLR activation with formulated (AtuPLEX), 2¶-O-methyl modified, research period. 23-mer, and blunt-ended siRNAs in vitro and after repeated systemic administration in mice (Supplementary Fig. S6). Beyond Acknowledgments the already published data (5, 13) on lack of IL-12p40 and IFN-a Received 6/25/2008; revised 8/27/2008; accepted 9/16/2008. stimulation, we expanded the panel of analyzed cytokines by IL-6, Grant support: Bundesministerium fu¨r Bildung und Forschung grant #0315156 IL-10, and tumor necrosis factor a. Again, no stimulation was PTJ. observed after systemic administration of Atu027 or an unrelated The costs of publication of this article were defrayed in part by the payment of page Luc charges. This article must therefore be hereby marked advertisement in accordance siRNA -AtuPLEX (Supplementary Fig. S6). Nevertheless, we have with 18 U.S.C. Section 1734 solely to indicate this fact. addressed the question on possible changes in levels of Kleinman We thank Dr Hu¨seyin Aygu¨n (BioSpring, Frankfurt am Main, Germany) for providing high-quality siRNA molecules, and Hans Meeldijk (Electron Microscopy, and colleagues’ (45) proposed ‘‘antiangiogenic acting’’ cytokines University of Utrecht, Utrecht, the Netherlands) for providing the electron microscopy IL-12 and IFN-g in our in vitro and in vivo settings (Supplementary picture.

References sterically stabilized nanoparticle. Nucleic Acids Res cells of different mouse tissues. Microvasc Res 2008;76: 2004;32:e149. 31–41. 1. Aagaard L, Rossi JJ. RNAi therapeutics: principles, 11. Pirollo KF, Rait A, Zhou Q, et al. Materializing the 19. Leenders F, Mopert K, Schmiedeknecht A, et al. prospects and challenges. Adv Drug Deliv Rev 2007;59: potential of small interfering RNA via a tumor-targeting PKN3 is required for malignant prostate cell growth 75–86. nanodelivery system. Cancer Res 2007;67:2938–43. downstream of activated PI 3-kinase. EMBO J 2004;23: 2. Aigner A. Applications of RNA interference: current 12. Li SD, Chen YC, Hackett MJ, Huang L. Tumor- 3303–13. state and prospects for siRNA-based strategies in vivo. targeted delivery of siRNA by self-assembled nano- 20. Czauderna F, Fechtner M, Dames S, et al. Structural Appl Microbiol Biotechnol 2007;76:9–21. particles. Mol Ther 2008;16:163–9. variations and stabilising modifications of synthetic 3. Uprichard SL. The therapeutic potential of RNA 13. Santel A, Aleku M, Keil O, et al. RNA interference in siRNAs in mammalian cells. Nucleic Acids Res 2003;31: interference. FEBS Lett 2005;579:5996–6007. the mouse vascular endothelium by systemic adminis- 2705–16. 4. Kim DH, Rossi JJ. Strategies for silencing human tration of siRNA-lipoplexes for cancer therapy. Gene 21. Stephenson RA, Dinney CP, Gohji K, Ordonez NG, disease using RNA interference. Nat Rev Genet 2007;8: Ther 2006;13:1360–70. Killion JJ, Fidler IJ. Metastatic model for human prostate 173–84. 14. Chien PY, Wang J, Carbonaro D, et al. Novel cationic cancer using orthotopic implantation in nude mice. 5. Santel A, Aleku M, Keil O, et al. A novel siRNA-lipoplex cardiolipin analogue-based liposome for efficient DNA J Natl Cancer Inst 1992;84:951–7. technology for RNA interference in the mouse vascular and small interfering RNA delivery in vitro and in vivo. 22. Fox SB, Harris AL. Histological quantitation of endothelium. Gene Ther 2006;13:1222–34. Cancer Gene Ther 2005;12:321–8. tumour angiogenesis. APMIS 2004;112:413–30. 6. Akhtar S, Benter IF. Nonviral delivery of synthetic 15. Landen CN, Jr., Chavez-Reyes A, Bucana C, et al. 23. Van der Auwera I, Cao Y, Tille JC, et al. First siRNAs in vivo. J Clin Invest 2007;117:3623–32. Therapeutic EphA2 gene targeting in vivo using neutral international consensus on the methodology of lym- 7. De Paula D, Bentley MV, Mahato RI. Hydrophobization liposomal small interfering RNA delivery. Cancer Res phangiogenesis quantification in solid human tumours. and bioconjugation for enhanced siRNA delivery and 2005;65:6910–8. Br J Cancer 2006;95:1611–25. targeting. RNA 2007;13:431–56. 16. Pal A, Ahmad A, Khan S, et al. Systemic delivery of 24. Morrissey DV, Lockridge JA, Shaw L, et al. Potent and 8. Li SD, Huang L. Gene therapy progress and prospects: RafsiRNA using cationic cardiolipin liposomes silences persistent in vivo anti-HBV activity of chemically non-viral gene therapy by systemic delivery. Gene Ther Raf-1 expression and inhibits tumor growth in xenograft modified siRNAs. Nat Biotechnol 2005;23:1002–7. 2006;13:1313–9. model of human prostate cancer. Int J Oncol 2005;26: 25. Duda DG, Fukumura D, Jain RK. Role of eNOS in 9. Hu-Lieskovan S, Heidel JD, Bartlett DW, Davis ME, 1087–91. neovascularization: NO for endothelial progenitor cells. Triche TJ. Sequence-specific knockdown of EWS-FLI1 by 17. Zhang C, Tang N, Liu X, Liang W, Xu W, Torchilin VP. Trends Mol Med 2004;10:143–5. targeted, nonviral delivery of small interfering RNA siRNA-containing liposomes modified with polyarginine 26. Zachary I. VEGF signalling: integration and multi- inhibits tumor growth in a murine model of metastatic effectively silence the targeted gene. J Control Release tasking in endothelial cell biology. Biochem Soc Trans Ewing’s sarcoma. Cancer Res 2005;65:8984–92. 2006;112:229–39. 2003;31:1171–7. 10. Schiffelers RM, Ansari A, Xu J, et al. Cancer siRNA 18. Aleku M, Fisch G, Mopert K, et al. Intracellular 27. Gupta GP, Massague J. Cancer metastasis: building a therapy by tumor selective delivery with ligand-targeted localization of lipoplexed siRNA in vascular endothelial framework. Cell 2006;127:679–95. www.aacrjournals.org 9797 Cancer Res 2008; 68: (23). December 1, 2008

28. Li J, Yen C, Liaw D, et al. PTEN, a putative protein 35. Alitalo K, Tammela T, Petrova TV. Lymphangio- 42. Gao D, Nolan DJ, Mellick AS, Bambino K, McDonnell tyrosine phosphatase gene mutated in human brain, genesis in development and human disease. Nature K, Mittal V. Endothelial progenitor cells control the breast, and prostate cancer. Science 1997;275:1943–7. 2005;438:946–53. angiogenic switch in mouse lung metastasis. Science 29. Hamada K, Sasaki T, Koni PA, et al. The PTEN/PI3K 36. Sundlisaeter E, Dicko A, Sakariassen PO, Sondenaa K, 2008;319:195–8. pathway governs normal vascular development and Enger PO, Bjerkvig R. Lymphangiogenesis in colorectal 43. Dirkx AE, Oude Egbrink MG, Wagstaff J, Griffioen tumor angiogenesis. Genes Dev 2005;19:2054–65. cancer-prognostic and therapeutic aspects. Int J Cancer AW. Monocyte/macrophage infiltration in tumors: 30. Graupera M, Guillermet-Guibert J, Foukas LC, et al. 2007;121:1401–9. modulators of angiogenesis. J Leukoc Biol 2006;80: Angiogenesis selectively requires the p110a isoform of 37. Thelen A, Scholz A, Benckert C, et al. Tumor- 1183–96. PI3K to control endothelial cell migration. Nature 2008; associated lymphangiogenesis correlates with lymph 44. Du R, Lu KV, Petritsch C, et al. HIF1a induces the 453:662–6. node metastases and prognosis in hilar cholangiocarci- recruitment of bone marrow-derived vascular modula- 31. Ceteci F, Ceteci S, Karreman C, et al. Disruption of noma. Ann Surg Oncol 2008;15:791–9. tory cells to regulate tumor angiogenesis and invasion. tumor cell adhesion promotes angiogenic switch and 38. Caunt M, Mak J, Liang WC, et al. Blocking neuropilin- Cancer Cell 2008;13:206–20. progression to micrometastasis in RAF-driven murine 2 function inhibits tumor cell metastasis. Cancer Cell 45. Kleinman ME, Yamada K, Takeda A, et al. Sequence- lung cancer. Cancer Cell 2007;12:145–59. 2008;13:331–42. and target-independent angiogenesis suppression by 32. Hanahan D, Folkman J. Patterns and emerging 39. Ferrara N, Hillan KJ, Gerber HP, Novotny W. siRNA via TLR3. Nature 2008;425:591–7. mechanisms of the angiogenic switch during tumori- Discovery and development of bevacizumab, an anti- 46. Judge AD, Bola G, Lee AC, MacLachlan I. Design genesis. Cell 1996;86:353–64. VEGF antibody for treating cancer. Nat Rev Drug Discov of noninflammatory synthetic siRNA mediating 33. Bergers G, Benjamin LE. Tumorigenesis and the 2004;3:391–400. potent gene silencing in vivo. Mol Ther 2006;13: angiogenic switch. Nat Rev Cancer 2003;3:401–10. 40. Ferrara N. Vascular endothelial growth factor as a 494–505. 34. Renyi-Vamos F, Tovari J, Fillinger J, et al. target for anticancer therapy. Oncologist 2004;9 Suppl 1: 47. Kim JY, Choung S, Lee EJ, Kim YJ, Choi YC. Lymphangiogenesis correlates with lymph node me- 2–10. Immune activation by siRNA/liposome complexes in tastasis, prognosis, and angiogenic phenotype in 41. Jain RK. Normalization of tumor vasculature: an mice is sequence-independent: lack of a role for human non-small cell lung cancer. Clin Cancer Res emerging concept in antiangiogenic therapy. Science Toll-like receptor 3 signaling. Mol Cells 2007;24: 2005;11:7344–53. 2005;307:58–62. 247–54.

Cancer Res 2008; 68: (23). December 1, 2008 9798 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2008 American Association for Cancer Research. Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Manuela Aleku, Petra Schulz, Oliver Keil, et al.

Cancer Res 2008;68:9788-9798.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/68/23/9788

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2008/11/25/68.23.9788.DC1

Cited articles This article cites 47 articles, 11 of which you can access for free at: http://cancerres.aacrjournals.org/content/68/23/9788.full#ref-list-1

Citing articles This article has been cited by 15 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/68/23/9788.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/68/23/9788. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.