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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.

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)

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

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.

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

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