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BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

Supplementary Figure 1 from BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells

Brain edema is a major neurological complication of acute liver failure (ALF) and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in this condition. Elevated brain ammonia level has been strongly implicated as an important factor in the mechanism of astrocyte swelling/brain edema in ALF. Recent studies, however, have suggested the possibility of a vasogenic component in the mechanism in ALF. We therefore examined the effect of ammonia on blood-brain barrier (BBB) integrity in an in vitro co-culture model of the BBB (consisting of primary cultures of rat brain endothelial cells and astrocytes). We found a minor degree of endothelial permeability to dextran fluorescein (16.2%) when the co-culture BBB model was exposed to a pathophysiological concentration of ammonia (5mM). By contrast, lipopolysaccharide (LPS), a molecule well-known to disrupt the BBB, resulted in an 87% increase in permeability. Since increased neurosteroid biosynthesis has been reported to occur in brain in ALF, and since neurosteroids are known to protect against BBB breakdown, we examined whether neurosteroids exerted any protective effect on the slight permeability of the BBB after exposure to ammonia. We found that a nanomolar concentration (10nM) of the neurosteroids allopregnanolone (THP) and tetrahydrodeoxycorticosterone (THDOC) significantly reduced the ammonia-induced increase in BBB permeability (69.13 and 58.64%, respectively). On the other hand, we found a marked disruption of the BBB when the co-culture model was exposed to the hepatotoxin azoxymethane (218.4%), but not with other liver toxins commonly used as models of ALF (thioacetamide and galactosamine, showed a 29.3 and 30.67% increase in permeability, respectively). Additionally, THP and THDOC reduced the effect of TAA and galactosamine on BBB permeability, while no BBB protective effect was observed following treatment with azoxymethane. These findings suggest that ammonia does not cause a significant BBB disruption, and that the BBB is intact in the TAA or galactosamine-induced animal models of ALF, likely due to the protective effect of neurosteroids that are synthesized in brain in the setting of ALF. However, caution should be exercised when using azoxymethane as an experimental model of ALF as it caused a severe breakdown of the BBB, and neurosteriods failed to protect against this breakdown.

Background. Interleukin receptor alpha 1 (IL-13RA1) is a component of a heterodimeric IL-13 receptor that is shared with IL-4 and it is ubiquitously expressed in normal tissues and also tumors. In contrast, IL-13RA2 is a monomeric receptor which is overexpressed in various solid tumors, but is virtually absent in normal tissues. Ligands specifically targeting IL-13RA2 but not IL-13RA1, such as Pep-1L and IL-13.E13K.D2.Cys, have been developed and validated [1,2]. Objective. To assess the expression of IL-13RA2 in pancreatic ductal adenocarcinoma and metastatic melanoma specimens and to develop tracers for selective recognition of the tumors. Methods. The expression of IL-13RA2in patient specimens, patient-derived xenografts (PDX) and established cell lines was assessed using Western blot, immunochemistry or immunofluorescence. The ligands were conjugated to a chelator and labeled with Yttrium 86. Ligand specificity was confirmed using IL-13RA2 positive and negative cells and blocking with a “cold” ligand. Biodistribution studies and PET/CT imaging were conducted to assess distribution of the tracer in IL-13RA2 positive tumors models. Results. IL-13RA2 receptor is overexpressed in melanoma and pancreatic cell lines and tumor tissues. Western blot analysis of 56 patients tumor tissue lysates obtained from pancreatic cancer PDX models showed the presence of IL-13RA2 in over 60% of patients. Similarly, tissue microarray analysis of metastatic melanoma samples identified IL-13RA2 receptor expression in 40% of tumor specimens. Uptake study performed in receptor positive and negative cells confirmed the specificity of radiotracer binding. In vivo experiments indicated fast clearance of 86Y- Pep1L ligand with glomerular filtration as a primary mechanism of the tracer removal. Mice bearing IL-13RA2 positive tumors had up taken the tracer with “tumor to muscle” ratio of 5.6 1 h after probe administration. Yttrium 86-labelled IL-13-E13K.D2.Cys was also taken up by IL-13 RA2 positive tumors. Conclusion. IL-13RA2 is an attractive target for the development of comprehensive theranostic strategies for melanoma and pancreatic cancer. References 1. Pandya, H., et al., An interleukin 13 receptor alpha 2-specific peptide homes to human Glioblastoma multiforme xenografts. Neuro Oncol, 2012. 14(1): p. 6-18. 2. Nguyen, V., et al., A novel ligand delivery system to non-invasively visualize and therapeutically exploit the IL13Ralpha2 tumor-restricted biomarker. Neuro Oncol, 2012. 14(10): p. 1239-53. Citation Format: Rafal Zielinski, Izabela Tworowska, Stanislaw Skora, Aleksandra Rusin, Radjendirane Venugopal, Arumugam Jayakumar, Izabela Fokt, Yaan Kang, Jason Fleming, Yue Huang, Denise Herpai, Waldemar Debinski, Waldemar Priebe. Targeting IL-13RA2 in melanoma and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1927.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is recognized as a serine protease inhibitor and is thought to play a key role in skin barrier function through the inhibition of kallikrein (KLK) activities and regulation of skin desquamation. LEKTI has a total of 15 potential inhibitory domains, and we hypothesize that it has other potential targets in the skin. To identify candidate protease targets of LEKTI, a label-free quantitative proteomic approach was employed. This work describes a novel, rapid, and noninvasive method for the identification and quantitation of the major proteins present in the uppermost layers of the skin. By using cells scraped from the elbow, we were able to rapidly identify and quantitate 79 proteins. Caspase 14 and bleomycin hydrolase were identified as the proteases of highest abundance. Despite the fact that caspase 14 is a cysteine protease and LEKTI is described as a serine protease inhibitor, we demonstrate that caspase 14 is inhibited by full-length LEKTI and 5 recombinant fragments of LEKTI to varied extents. Details of the development of the methods used for the creation of the skin proteome and the inhibition of caspase 14 by LEKTI and implications for LEKTI as a multifunctional protease inhibitor are discussed.

Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA). We then examined the activity of Ca2+-dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS), and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pre-treatment of cultures with 7-nitroindazole, apocyanin, and quinacrine, respective inhibitors of cNOS, NOX, and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX, and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling.

Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1–6 (rLEKTI(1–6)), domains 6–8 and partial domain 9 (rLEKTI(6–9′)), domains 9–12 (rLEKTI(9–12)), and domains 12–15 (rLEKTI(12–15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1–6), rLEKTI(6–9′), and rLEKTI(9–12) with Ki values in the range of 2.3–28.4 nm, 6.1–221 nm, and 2.7–416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12–15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.

Here, we review the literature on the molecular and biochemical properties of Lympho-Epithelial Kazal-Type-Inhibitor (LEKTI). Serine Protease Inhibitor Kazal- type 5 (SPINK5) gene encodes three different LEKTI isoforms. These isoforms are organized into a typical 15, longer than 15 and shorter than 15 inhibitory domains consisting of 1064, 1094 and 916 residues respectively. LEKTI isoforms synthesized as pro-LEKTI proteins are processed intracellular and secreted as bioactive LEKTI fragments into blood. LEKTI potently inhibits activity of plasmin, subtilisin A, cathepsin G, elastase, trypsin, kallikrein (KLK) 5, KLK6, KLK7, KLK13, and KLK14 to varied extents. Mutations in SPINK5 gene resulting in decreased LEKTI function and increased KLK activity causes Netherton syndrome (NS). Low SPINK5 expression/LEKTI function is also associated with Head and Neck Squamous Cell carcinoma (HNSCC), chronic rhinosinusitis and asthma. Thus restoring SPINK5 expression/LEKTI function in NS, HNSCC and asthma holds therapeutic promise.

Headpin is a novel serine proteinase inhibitor (serpin) with constitutive mRNA expression in histologically normal oral mucosa but with lost or down-regulated expression in head and neck squamous cell carcinoma. Several serpin family members are similarly lost in multiple cancer types and hold tumor suppressor functions including the inhibition of angiogenesis. However, the functional significance for the loss of headpin expression in cancer is not known. Using immunohistochemical analysis of invasive squamous cell carcinoma and matched normal squamous mucosa of patient specimens, headpin expression was lost or down-regulated in the vast majority of tumor specimens. We investigated the functions of exogenous recombinant headpin and endogenously expressed headpin related to angiogenesis. In a rat corneal assay of neovascularization, recombinant headpin protein blocked in vivo angiogenesis mediated by interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF). In assays of cellular events in angiogenesis, headpin blocked the invasion, migration, and tube formation of endothelial cells. In light of our findings of nuclear subcellular localization of headpin, we investigated the expression and secretion of angiogenic factors and found reduced mRNA, protein, and promoter activities of IL-8 and VEGF. Finally, using a murine flank tumor model, headpin expression reduced growth and microvessel density in tumors derived from headpin-expressing UMSCC1 cells relative to those from vector control cells. These findings of nuclear regulatory functions of a serpin in the inhibition of angiogenesis bring new understanding to the cellular and molecular mechanisms of serpins. Therefore, this novel serpin targets diverse mechanisms against tumor angiogenesis on which to base therapeutic strategies. (Cancer Res 2005; 65(24): 11501-9)

The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60 kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion.

The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9' was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9' (rLEKTI6-9') with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9' inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9' occurred through a noncompetitive mechanism, with inhibitory constants (Ki) of 356 +/- 12 and 193 +/- 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9' was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9', recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 +/- 10 nM. Taking LEKTI6-9' as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.

The human LEKTI gene encodes a putative 15-domain serine proteinase inhibitor and has been linked to the inherited disorder known as Netherton syndrome. In this study, human recombinant LEKTI (rLEKTI) was purified using a baculovirus/insect cell expression system, and the inhibitory profile of the full-length rLEKTI protein was examined. Expression of LEKTI in Sf9 cells showed the presence of disulfide bonds, suggesting the maintenance of the tertiary protein structure. rLEKTI inhibited the serine proteinases plasmin, subtilisin A, cathepsin G, human neutrophil elastase, and trypsin, but not chymotrypsin. Moreover, rLEKTI did not inhibit the cysteine proteinase papain or cathepsin K, L, or S. Further, rLEKTI inhibitory activity was inactivated by treatment with 20 mM DTT, suggesting that disulfide bonds are important to LEKTI function. The inhibition of plasmin, subtilisin A, cathepsin G, elastase, and trypsin by rLEKTI occurred through a noncompetitive-type mechanism, with inhibitory constants (K(i)) of 27 +/- 5, 49 +/- 3, 67 +/- 6, 317 +/-36, and 849 +/- 55 nM, respectively. Thus, LEKTI is likely to be a major physiological inhibitor of multiple serine proteinases.

Headpin (SERPINB13) is a novel member of the serine proteinase inhibitor (Serpin) gene family that was originally cloned from a keratinocyte cDNA library. Western blot analysis using a headpin-specific antiserum recognized a protein with the predicted M(r) of 44kDa in lysates derived from a transformed keratinocyte cell line known to express headpin mRNA. Similarity of the reactive-site loop (RSL) domain of headpin, notably at the P1-P1(') residues, with other serpins that inhibit cysteine and serine proteinases suggests that headpin may inhibit similar proteinases. This study demonstrates that recombinant headpin indeed inhibits cathepsins K and L, but not chymotrypsin, elastase, trypsin, subtilisin A, urokinase-type plasminogen activator, plasmin, or thrombin. The second-order rate constants (k(a)) for the inhibitory reactions of rHeadpin with cathepsins K and L were 5.1+/-0.6x10(4) and 4.1+/-0.8x10(4)M(-1)s(-1), respectively. Headpin formed SDS-stable complexes with cathepsins K and L, a characteristic property of inhibitory serpins. Interactions of the RSL domain of headpin with cathepsins K and L were indicated by cleavage of headpin near the predicted P1-P1(') residues by these proteinases. These results demonstrate that the serpin headpin possesses specificity for inhibiting lysosomal cysteine proteinases.

Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different Lympho- Epithelial Kazal-Type-Inhibitor (LEKTI) isoforms which are organized into longer than 15, 15, and 13 inhibitory domains. We identified LEKTI by its constitutive expression in normal oral mucosa and lost or down regulated expression in matched tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Previously, we showed that recombinant full-length LEKTI and rLEKTI fragments inhibit the activity of plasmin, subtilisin A, cathepsin G, neutrophil elastase, trypsin, caspase 14, and kallikreins (KLK) 5, 6, 7, 13, and 14 to varied extents. Here, we show that LEKTI protein is absent in HNSCC OSC19, Tu138, Tu177, and UMSCC1 lines. We then determined the consequences of LEKTI re-expression on migration and invasion, adhesion and gene expression profile of HNSCCOSC19 and UMSCC1 lines. We demonstrate that LEKTI expressing OSC19 and UMSCC1 clones show markedly reduced migration and invasion. Moreover, LEKTI expressing OSC19 clones show striking morphological changes and enhanced adhesionon type I, III, IV, and V collagens, fibronectin, and laminin5.In addition, we show that exogenous r-LEKTI blocks migration of OSC19-parental cells in a dose and time dependent manner. Microarray analysis identified 186 genes which are differentially regulated in both OSC19 LEKTI clones. MMP-14, KLK5, and ADAM8 are down regulated while MMP-3, LEKTI, DSC2 and DSC3are up-regulated in OSC19 LEKTI clones. RT-PCR and Western blot results confirmed microarray results for MMP-14 and MMP-3in OSC19 LEKTI clones. In addition we discover that MMP-9 protein expression and pro-MMP-9 activity are severely reduced in LEKTI expressing clones as shown by WB and zymogram. Together, this work provides mechanistic insights into how loss of LEKTI protein expression promotes an invasive phenotype in HNSCC tumors.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes., Cellular and Molecular Life Sciences, 70 (2), ISSN:1420-682X, ISSN:1420-9071

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

We have cloned and sequenced the cDNA coding for human HepG2 acetyl-CoA carboxylase (ACC; EC 6.4.1.2). The sequence has an open reading frame of 7038 bp that encode 2346 amino acids (M(r), 264,737). The C-terminal 2.6-kb sequence is very different from that recently reported for human ACC (Ha, J., Daniel, S., Kong, I.-S., Park, C.-K., Tae, H.-J. & Kim, K.-H. [1994] Eur. J. Biochem. 219, 297-306). Northern blot analysis revealed that the ACC mRNA is approximately 10 kb in size and that its level varies among the tissues tested. Evidence is presented to show that the human ACC gene is 200-480 kbp in size and maps to chromosome 17q12. We also provide evidence for the presence of another ACC-like gene with similarly sized mRNA but tissue-specific expression different from that of the ACC gene reported herein. That this second ACC-like gene encodes the 280-kDa carboxylase is not ruled out.

Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI) has been demonstrated to be an inhibitor of various kallikreins and is thought to play a role in the regulation of skin desquamation. In order to identify and investigate the potential of LEKTI to interact with other proteins, a method was developed using immobilised proteins onto arrays and nanoUPLC/MALDI-TOF MS. Using various domains of LEKTI, we demonstrated that these domains bound a number of kallikreins (5, 13 and 14) to varied extents on the array surface. Inhibitory assays confirmed that binding on the protein array surface corresponded directly to levels of inhibition. The method was then tested using skin epidermal extracts. All forms of rLEKTI with the exception of rLEKTI 12-15, demonstrated the binding of several potential candidate proteins. Surprisingly, the major binding partners of LEKTI were found to be the antimicrobial peptide dermcidin and the serine protease cathepsin G and no kallikreins. Using confocal microscopy and Netherton syndrome skin sections, we confirmed the co-localisation of LEKTI with dermcidin and demonstrated altered trafficking of dermcidin in these patients. This potential new role for LEKTI as a multifunctional protein in the protection and transport of proteins in the epidermis and its role in disease are discussed.

We have isolated and sequenced 0.5- and 3.6-kb cDNA clones that cover the N-terminal and carboxy-terminal regions, respectively, of the human fatty acid synthase. To localize the fatty acid synthase gene and to define its genomic structure, we have also isolated overlapping genomic clones by screening two human YAC libraries with PCR primers derived from the fatty acid synthase cDNA sequences. The DNA inserts in these human fatty acid synthase YACs hybridized with human synthase-specific cDNA probes. Using biotin-labeled Alu-PCR products of the human synthase YACs as probes for fluorescence in situ hybridization, we mapped the fatty acid synthase gene to chromosome 17q25. We also screened a chromosome 17-specific cosmid library with human synthase DNA probes and isolated 12 cosmids, all of which had EcoRI fragments in common. DNA sequencing of an amplified PCR product from the fatty acid synthase cosmids confirmed that these genomic clones contained expressed fatty acid synthase sequences. Furthermore, the results of Southern analyses suggested that a single 40-kb cosmid clone encompasses the entire coding region of the fatty acid synthase gene. The synthase gene is located on chromosome 17 near the q25 band, which is close to the telomere and could serve as an important marker in analysis of this chromosome.

Purpose: Melanoma is a common and deadly tumor that upon metastasis to the central nervous system has a median survival duration of Experimental Design: Combinational therapy of STAT3 blockade agents with IFN-α was investigated in a metastatic and an established syngeneic intracerebral murine tumor model of melanoma. The immunologic in vivo mechanisms of efficacy were investigated by T-cell and natural killer (NK) cell cytotoxic assays.Results: IFN-α immunotherapy was synergistic with WP1193 showing marked in vivo efficacy against metastatic and established intracerebral melanoma. At autopsy, it was noted that there was a decreased trend in mice with melanoma developing leptomeningeal disease treated with combinational therapy. The combinational approach enhanced both NK-mediated and T-cell–mediated antitumor cytotoxicity.Conclusions: The immune modulatory effects of STAT3 blockade can enhance the therapeutic efficacy of IFN-α immunotherapy by enhancing both innate and adaptive cytotoxic T-cell activities. This combination therapy has the potential in the treatment of metastatic melanoma that is typically refractory to this type of immune therapeutic approach. Clin Cancer Res; 16(9); 2550–61. ©2010 AACR.

The oncogenic role of STAT3 has been elucidated in a number of human malignancies including leukemia, lymphoma, malignant glioma and cancers of the breast, lung, and head and neck (HNSCC). Here we show that WP1066 has profound anti-neoplastic effects in HNSCC, mediated in part by suppression of JAK2-STAT3 signaling. WP1066 inhibited constitutive and inducible STAT3 phosphorylation in both dose- and time-dependant manners. Further, the nuclear translocation of STAT3 was completely inhibited, resulting in decreased DNA binding activity. In vivo testing of WP1066 in a nude mouse orthotopic model of HNSCC demonstrated significant anti-tumor effects, with histological evidence of decreased cellular proliferation and angiogenesis. Collectively, these data suggest that WP1066 suppresses squamous cell carcinoma cell growth, in part through its effects on JAK-STAT pathways, and establishes this small molecule as potentially efficacious agent in the treatment of HNSCC.

LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8–D11, and D9–D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8–D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.

Melanoma is expected to be diagnosed in 76,100 patients in the United States, and 9,710 patients are estimated to die of the disease in 2014. Early-stage melanomas can often be treated effectively with surgery alone, but more advanced cancers often require other treatments, including radiation therapy, immunotherapy, chemotherapy and targeted therapy. Targeted therapy with vemurafenib is used to treat approximately 60 percent of melanoma patients with activating mutation in the BRAF gene. V600E mutation in the BRAF gene is detected in over half of the melanoma patients and targeted inhibition of the oncogenic BRAF has shown a very good initial response in melanoma patients, however, this treatment alone turned out to be unsuccessful due to the rapid development of resistance. Development of therapeutics for use in combination with BRAF inhibitors or strategies overcoming the resistance to vemurafenib is a subject of intensive research. Systematic screening of unique DNA bisintercalating agents of our own design using NCI 60 cell line panel led to identification of highly potent and melanoma selective compound WP760. The main aim of this study was to assess antiproliferative activity of WP760 in a panel of melanoma cell lines including BRAFi resistant cell lines. Here we have shown that WP760 inhibited proliferation of melanoma cell in low nanomolar range and was not toxic to normal fibroblasts. Importantly, WP760 inhibited potently proliferation of cells resistant to BRAF inhibitors and wild type BRAF in low nanomolar range. Moreover, it was equally active in pairs of isogenic cell lines: naïve and those which developed resistance to vemurafenib. The second aim of this work was preliminary assessment of WP760 toxicity in vivo. In our first attempt to perform in vivo characterization of WP760, we analyzed its solubility and developed formulation in 5% dextrose in a presence of 5% DMSO suitable for intravenous administration. Our preliminary toxicity study performed in CD-1 mice, indicated low or no apparent toxicity symptoms when WP760 was dosed up to 10 mg/kg (rate of death 14%). For multiple dosing, when animals received three IV injections of WP760, the dose of 2.5 mg/kg appeared to be safe and animals did not present abnormal behavior when observed over 30 days period. Mice receiving higher doses of the compound developed toxicity symptoms after 3rd dose and the time they appeared was dose dependent. The observed melanoma selectivity of WP760 and its low nanomolar range cytotoxic potency in vitro combined with the lack of toxicity in normal fibroblasts were in agreement with results of the in vivo experiments demonstrating relatively high maximum tolerated dose in animals suggesting on the existence of a broad therapeutic window in vivo. Acknowledgment: This research was supported in part by funds from the University Cancer Foundation via the Sister Institution Network Fund at the UT MD Anderson Cancer Center and Melanoma SPORE (2 P50 CA093459) Citation Format: Aleksandra Rusin, Rafal Zielinski, Izabela Fokt, Van Nguyen, Stanislaw Skora, Arumugam Jayakumar, Waldemar Priebe. WP760, A new highly potent and selective agent against melanoma including BRAFi resistant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4473. doi:10.1158/1538-7445.AM2015-4473

Background: Our drug discovery program has led to the development of a series of diverse, unique small molecule drugs targeting the key oncogenic transcription factor STAT3 which drives diverse human cancers. WP1066 was selected as a lead drug because of its drug-like properties, therapeutic window and validated in vivo activity in a broad range of tumor models. During the process of WP1066 development as a clinical phase new agent, we developed and further characterized an orally bioavailable formulation of WP1066 using a spray dried oral formulation. Methods: The spray dried formulation of WP1066 (SDD1) was prepared using Hypermellose acetate succinate (HPMCAS) as an enteric coating film. HPMCAS is a mixture of acetic and monosuccinic acid esters of hydroxypropylmethyl cellulose. While insoluble in gastric fluids, the SDD1 formulation swells and leads to rapid dissolution in the higher pH found in upper small intestine, and subsequent release of the drug. For theses studies SDD1 was characterized for size, stability and purity. Single and multiple-dose toxicity studies were done in female CD-1 mice, using 5% dextrose as a vehicle. Single dose PK and organ distribution analysis was performed after oral gavage administration of 200 mg/kg after 12 hour fasting. Animals were sacrifice at different time-points (2 min to 24 hours). Plasma, brain and pancreas were isolated and the apparent concentration of WP1066 was determined using HPLC/MS/MS analysis. For multi-dose PK analysis, animals were fasted for six hours, dosed with SDD1 at 200 mg/kg. Animals were euthanized at different time intervals after the second dose (given six hours after first dose). Results: Mice dosed with SDD1 did not show any apparent toxicity when administered up to 200 mg/kg. For multiple doses, mice received 50, 100 or 200 mg/kg of WP1066 in SDD1 every day for 28 days. No apparent toxicity or significant weight lost was observed. In a similar way, animals dosed twice a day with 200 mg/kg of WP1066 (SDD1) for 15 doses did not show any symptoms of toxicity. Pilot PK analysis confirmed dose-dependent absorption of WP1066. Single-dose PK analysis revealed rapid GI absorption with a TMAX = 45 min and CMAX in plasma 1.5 μg/ml (which corresponds to ∼4 μM). The estimated half-life was approximately 3 hrs. Similar parameters were obtained from PK analysis performed in canines. SDD1 dosing led to significant accumulation of WP1066 in intact brain and pancreatic tissues in mice. These results support further preclinical development of SDD1 formulated WP1066 aimed the initiation of clinical studies in pancreatic, brain cancer and melanoma metastasis to brain patients. Acknowledgment: These studies were supported in part by the grants: Brain SPORE (2 P50 CA127001), Melanoma SPORE (2 P50 CA093459); Viragh Foundation, Houston Pharmaceuticals. Citation Format: Rafal Zielinski, Aleksandra Rusin, Timothy Madden, Charles Conrad, Mary Johansen, Izabela Fokt, Stanislaw Skora, Arumugam Jayakumar, Amy Heimberger, Waldemar Priebe. Development of orally bioavailable formulation of WP1066 and its evaluation in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4540. doi:10.1158/1538-7445.AM2015-4540

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of diseases characterized by clonal expansion of malignant T-cells in the skin. In the course of our quest to screen and identify a potent, selective anti-proliferative drug for CTCL, we discovered that caffeic acid benzyl ester (CABE), a natural component of edible plant species found in high concentrations in propolis, showed increased potency for CTCL (IC50 4 μM) in vitro compared to other tumor types such as melanoma (IC50 25 μM), glioma (IC50 25 μM), multiple myeloma (MM) (IC50 70 μM), head and neck squamous cell carcinoma (HNSCC) (IC50 25 μM), ovarian cancer (OC) (IC50 17 μM), breast carcinoma (IC50 43 μM), and Hodgkin's B-cell lymphoma (IC50 33 μM). Cell death in CTCL MJ and HH cell lines was accompanied by increased caspase-3 and poly (ADP-ribose) (PARP) cleavage and annexin V staining. There is growing evidence that CABE and its analogs exert their anticancer activity by modulating expression level of key transcription factors. Here, we evaluated the effects of CABE on the activity of different transcription factors and the expression of total and phosphorylated proteins in MJ cells by use of the transcription factor activation profiling array (TFAPA) and the reverese-phase protein array profiling (RPPA) assays. The activity of 48 transcription factors (TFs) and expression of 225 unique total and phosphorylated proteins could be monitored simultaneously by using a collection of biotin-labeled DNA probes and 225 unique antibodies. TFAPA analysis revealed that 18 TFs (including STAT1, STAT4, and RXR) were up-regulated and 3 TFs (STAT3, STAT5, and SATB1) were down-regulated to a variable extent in cells treated with 10 μM CABE for 5 h as compared to vehicle-treated control. RPPA analysis revealed 41 differentially expressed proteins between vehicle control and cells treated with 10 μM CABE. These can be functionally categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. Our results suggest that CABE targets multiple signaling pathways in CTCL in vitro, warranting the development of a pre-clinical animal model for the testing of CABE efficacy in vivo. Acknowledgement: These studies were supported in part by the grant from Moleculin, LLC Citation Format: Arumugam Jayakumar, Radjendirane Venugopal, Rafal Zielinski, Aleksandra Rusin, Izabela Fokt, Stanislaw Skora, Xiao Ni, Madeleine Duvic, Waldemar Priebe. High sensitivity of cutaneous T-cell lymphoma (CTCL) to CABE, a component of propolis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2015-2096

Background Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro . Methods Expression of 12 of the KLKs ( KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities. Results We here describe for the first time expression of numerous KLKs ( KLKs 5–8, 10–14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4–6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro . Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro . Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation. Conclusion We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.

Steroid hormones regulate a wide range of physiologic functions in humans. The cholesterol side-chain cleavage enzyme P450scc regulates the initial step of biosynthesis of all steroid hormones. We investigated the expression of P450scc by studying a potential regulator of P450scc, LBP-32/MGR. Using a Northern blot, we found that LBP-32/MGR mRNA was expressed mainly in the human placenta. Using radiation hybrid mapping, we identified LBP-32/MGR on human chromosome 2p25. Recombinant LBP-32/MGR protein bound preferentially to a DNA fragment from the promoter of P450scc in vitro and exhibited clear nuclear localization in transfected cells. Luciferase reporter gene assays showed that LBP-32/MGR specifically repressed transcriptional activation of the human P450scc promoter. Because placental P450scc expression is essential for pregnancy and steroid biosynthesis, the placental expression and transcriptional repressor activity of LBP-32/MGR in JEG-3 cells suggest it has a role as a transcriptional modulator of steroid biosynthesis.

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., Kd, ∼2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

Headpin is a novel serine proteinase inhibitor (serpin) that is downregulated in many established HNSCC tumor cell lines and human oral SCC specimens. The use of the bacterial and yeast expression systems for headpin resulted in poor yields and proteins with low inhibitory activity. To circumvent these problems, we have developed a baculovirus-insect cell system for high-yield expression as well as fully functional protein. Here, we describe the strategies and methods used to express headpin in an insect cell heterologous system. In addition, procedures to purify the recombinant proteins are described. A metal affinity column followed by a gel-filtration chromatography provides a rapid and efficient method for large quantity preparation of headpin. This method should be useful as an alternative expression system for those serpins that are not purifiable when expressed using the Escherichia coli or yeast expression system.

Rituximab in combination with hyperfractionated cyclophosphamide, vincristine, doxorubicin (DOX), and dexamethasone has been a successful frontline therapy for aggressive mantle cell lymphoma (MCL), a distinct and heterogeneous subtype of non-Hodgkin lymphoma, with complete remission rates as high as 87% in patients at or below 65 years of age. However, the efficacy of rituximab in combination with hyperfractionated cyclophosphamide, vincristine, DOX, and dexamethasone comes at the expense of severe grade III-IV toxic effects, and its use is limited by de novoor acquired multidrug resistance. Berubicin (WP744; BRN) is a clinically evaluated analog of DOX that, like DOX, stabilizes cleavable complexes between DNA and topoisomerase II, leading to irreversible double-strand DNA breaks and abrogation of DNA religation. In contrast to DOX, BRN overcomes multidrug resistance by circumventing efflux mediated by ATP-binding cassette proteins like P-glycoprotein and MRP-1. BRN is also the first anthracycline derivative to effectively cross the blood-brain barrier, with demonstrated phase 1 responses in brain cancer patients. Our MCL studies demonstrated that treatment with BRN decreased cell growth threefold to sixfold more potently than DOX in all tested MCL cell lines (Mino, Jeko-1, SP53, and Granta 519) and four MCL patient samples by arresting MCL cell-cycle progression at the G2/M phase and inducing extensive apoptosis. Moreover, in a Western blot assay, Jeko-1 and Mino cells exhibited activation of caspase-3 and caspase-9 and cleavage of poly(ADP-ribose) polymerase over time, pointing to the presence of a mitochondria-mediated apoptosis pathway in BRN-treated MCL cell lines. We then assessed the in vivo activity of BRN, using DOX as a control, in MCL-bearing severe combined immunodeficient (SCID) and SCID-hu mice. Our results indicated that SCID mice given 10 mg/kg DOX survived no more than 15 days after treatment, whereas all mice given 10 mg/kg BRN survived up to 63 days after treatment. On the other hand, when BRN and DOX were administered at lower doses of 5 mg/kg in a SCID-hu MCL mouse model, similar survival durations for BRN and DOX were observed. In summary, our studies demonstrate that BRN is highly potent against MCL in vitro and displays a wider therapeutic window than DOX, delivering very promising in vivo activity in both SCID and SCID-hu MCL models. These results indicate that BRN should be evaluated in the treatment of other lymphomas and seriously considered as an effective replacement for DOX in MCL treatment. BRN’s success in Phase 1 studies in glioma patients and observed safety and effectiveness in MCL models warrant further investigation of its clinical use and combination with existing therapies in MCL patients. Disclosures Priebe: MD Anderson: Waldemar Priebe holds patents and has a financial interest in the development of berubicin. Patents & Royalties.

BACKGROUND: Pancreatic cancer (PC) is one of the most deadly forms of human cancer with 5-year survival less than 5%. Presently the nucleoside analog gemcitabine is used for front line disease management but its low response rate and the frequent development of drug resistance, which is poorly understood, limits its effectiveness. A large body of literature indicates that the STAT3, STAT5, and NF-κB pathways are active drivers of pancreatic cancer development and progression. Agents targeting STAT3 and NF-κB pathways are being actively being considered as potentially effective drugs and have been tested in in vitro and in vivo pancreatic tumor models with limited success. EXPERIMENTAL DESIGN: We aimed to reassess the role and function of these three transcription factors in a panel of PC cell lines and assess the activity of WP1066, our lead STAT3/STAT5 inhibitor, and its close congeners in the same lines. Our reevaluation studies of the oncogenic function of STAT3, STAT5 and NF-κB were carried out in 3 established pancreatic cancer cell lines (PANC-1, Colo357, and MIAPaCa2) and 2 primary tumor cell lines (MDAPATC53 and MDAPATC50) isolated from patient tumor specimens with known clinical features and available relevant in vivo models. PC cells were separately treated with cytokines (IL-6 and IFN-α), gemcitabine and TNF-α and fractionated into cytoplasmic and nuclear portions. Levels of p-STAT3, p-STAT5, and NF-κB were assessed using Western blot, MesoScaleDiscovery, DNA binding activity, and confocal methods. RESULTS: Collectively, our data showed that STAT3 was constitutively activated and that tyrosine-phosphorylated STAT3 (p-STAT3Y705) is exclusively located in the nucleus of the ColoFG357 and MiaPaCa2 cell lines. IL-6 and IFN-α treatment induced p-STAT3Y705, and their combined use resulted in synergistically higher levels of p-STAT3Y705 and in nuclear localization in all five PC cell lines. In contrast, and rather surprisingly, the tyrosine-phosphorylated STAT5Y694 and NF-κB were mostly localized in the cytoplasm in all five cell lines. Consistent with these results, the DNA binding activity of p-STAT5 and NF-κB is very low compared to p-STAT3. Interestingly, our studies revealed that gemcitabine treatment triggers the nuclear accumulation of NF-κB in a time-dependent manner suggesting that a combination of gemcitabine with NF-κB inhibitors can have synergistic activity in PDAC. CONCLUSION: Our findings contribute to unraveling the functional significance of p-STAT3 and NF-κB as oncogenic transcription factors during pre- and post- gemcitabine treatment and the critical need to concurrently inhibit several targets and develop drug combinations or multitargeted drugs that block these pathways. Acknowledgement: This research was supported by the grant from Viragh's Foundation. Citation Format: Arumugam Jayakumar, Venugopal Radjendirane, Jason Fleming, Yaan Kang, Aleksandra Rusin, Rafal Zielinski, Stanislaw Skora, Izabela Fokt, Waldemar Priebe. Status of STAT3, STAT5, and NF-κB in pancreatic cancer cell lines, small molecule inhibitors, and potential clinical implications. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2946. doi:10.1158/1538-7445.AM2014-2946