Arumugam Jayakumar

A POTENTIAL ROLE FOR MULTIPLE TISSUE KALLIKREIN SERINE PROTEASES IN EPIDERMAL DESQUAMATION

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.

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

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.

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI).

Abstract LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6–8 and 9–12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (K i) measured either directly in concentration-dependent studies or calculated from measured association (k ass) and dissociation (k dis) rate constants were 1.2–5.5 nM at pH 8.0 and 10–20 nM at pH 5.0. At pH 8.0, k ass and k dis values were 4.7×105 M−1 s−1 and 5.5×10−4 s−1, and at pH 5.0 they were 4.0×104 M−1 s−1 and 4.3×10−4 s−1, respectively. The low K i and k dis values (t 1/2 of 20–25 min) indicate tight and specific association. Only fragment 6–9′ was a good inhibitor of rhK7, demonstrating a K i of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.

Inhibition of p-STAT3 Enhances IFN-α Efficacy against Metastatic Melanoma in a Murine Model

Purpose: Melanoma is a common and deadly tumor that upon metastasis to the central nervous system has a median survival duration of <6 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. We hypothesized that WP1193, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of IFN-α against metastatic melanoma. 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.

Headpin: A Serpin with Endogenous and Exogenous Suppression of Angiogenesis

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.

The identification of a new role for LEKTI in the skin: The use of protein ‘bait’ arrays to detect defective trafficking of dermcidin in the skin of patients with Netherton syndrome

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.

Production of serpins using baculovirus expression systems.

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.

Abstract 3251: Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC WP1066 was shown to exert its potent inhibitory activity against several types of human cancer including melanoma in vitro and in vivo via suppression of JAK2/STAT3/STAT5 activation. However, if there is any other additional molecular mechanism(s) by which WP1066 generates its anti-tumor effects remain largely unknown. Here, we report a novel function for WP1066 in inducing the degradation of both HIF-1α and HIF-1β subunits in human melanoma cells. We found constitutive activation of HIF-1α and an increased expression of hexokinase II, a down target of HIF-1α, in normoxia in 6/6 of melanoma cell lines, but not in other tumor types. Both hypoxia and cobalt chloride, a transition metal that mimics hypoxia, further increased HIF-1α protein accumulation in a time-dependent manner in WM35 and SKMEL-1 melanoma cells. WP1066 selectively decreased the constitutive and hypoxia and cobalt chloride induced increase in the level of HIF-1α in a dose-dependent manner with a half-life (T1/2) of ∼13, ∼10, and ∼10 min in WM35 cells. Moreover, WP1066 decreased the T1/2 of HIF-1α following cycloheximide treatment from ∼13 to ∼7 min suggesting that WP1066 induced HIF-1α instability is not mediated via inhibition of its translation. Further, inhibition of HIF-1α protein accumulation by WP1066 was not abolished in the presence of MG132 and proteasome inhibitor I, a potent inhibitor of the 26S proteasome, suggesting that the 26S proteasome-system is not responsible for WP1066-induced HIF-1α instability. Immunoprecipitation, confocal microscopy, and cellular fractionation analyses revealed that WP1066 induced ubiquitinylation, aggregation and degradation of HIF-1α in WM35 cells. Finally, WP1066 directly inhibits activity of rhUSP5 and rUCH-L1. Altogether, our study identified a novel function for WP1066 in inducing HIF-1 instability that may also contribute to the anti-cancer effects of WP1066 in human melanoma. Citation Format: Arumugam Jayakumar, Jana Rauvolfova, Hanying Bao, Izabela Fokt, Stanislaw Skora, Amy Heimberger, Waldemar Priebe. Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2013-3251

Abstract 4540: Development of orally bioavailable formulation of WP1066 and its evaluation in vivo

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

Abstract 2946: Status of STAT3, STAT5, and NF-κB in pancreatic cancer cell lines, small molecule inhibitors, and potential clinical implications

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-STAT3Y 705 ) is exclusively located in the nucleus of the ColoFG357 and MiaPaCa2 cell lines. IL-6 and IFN-α treatment induced p-STAT3Y 705 , and their combined use resulted in synergistically higher levels of p-STAT3Y 705 and in nuclear localization in all five PC cell lines. In contrast, and rather surprisingly, the tyrosine-phosphorylated STAT5Y 694 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 Viragh9s 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