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RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of β-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on β-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2–M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear β-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFβ, and Wnt/β-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear β-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

Survival curves for RX-5902 treatment and Abraxane and Vehicle control in vivo

Introduction: RX-3117 is an oral, small molecule cytidine analog anticancer agent with an improved pharmacological profile relative to gemcitabine and other nucleoside analogs. The agent has excellent activity against various cancer cell lines and xenografts including gemcitabine-resistant variants and it has excellent oral bioavailability; it is not a substrate for the degradation enzyme cytidine deaminase. RX-3117 is being evaluated at a daily oral schedule of 700 mg (5 days/week for 3 weeks) which results in plasma levels in the micromolar range that have been shown to be cytotoxic to cancer cells. It has shown clinical activity in refractory bladder cancer and pancreatic cancer. Areas covered: The review provides an overview of the relevant market and describes the mechanism of action, main pharmacokinetic/pharmacodynamic features and clinical development of this investigational small molecule. Expert opinion: RX-3117 is selectively activated by uridine-cytidine kinase 2 (UCK2), which is expressed only in tumors and has a dual mechanism of action: DNA damage and inhibition of DNA methyltransferase 1 (DNMT1). Because of its tumor selective activation, novel mechanism of action, excellent oral bioavailability and candidate biomarkers for patient selection, RX-3117 has the potential to replace gemcitabine in the treatment of a spectrum of cancer types.

Background Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC. Methods Treatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells. Results The addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p p Conclusions Conclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

(1) Background: RX-3117 (fluorocyclopentenyl-cytosine) is a cytidine analog that inhibits DNA methyltransferase 1 (DNMT1). We investigated the mechanism and potential of RX-3117 as a demethylating agent in several in vitro models. (2) Methods: we used western blotting to measure expression of several proteins known to be down-regulated by DNA methylation: O6-methylguanine-DNA methyltransferase (MGMT) and the tumor-suppressor genes, p16 and E-cadherin. Transport of methotrexate (MTX) mediated by the proton-coupled folate transporter (PCFT) was used as a functional assay. (3) Results: RX-3117 treatment decreased total DNA-cytosine-methylation in A549 non-small cell lung cancer (NSCLC) cells, and induced protein expression of MGMT, p16 and E-cadherin in A549 and SW1573 NSCLC cells. Leukemic CCRF-CEM cells and the MTX-resistant variant (CEM/MTX, with a deficient reduced folate carrier) have a very low expression of PCFT due to promoter hypermethylation. In CEM/MTX cells, pre-treatment with RX-3117 increased PCFT-mediated MTX uptake 8-fold, and in CEM cells 4-fold. With the reference hypomethylating agent 5-aza-2&prime, deoxycytidine similar values were obtained. RX-3117 also increased PCFT gene expression and PCFT protein. (4) Conclusion: RX-3117 down-regulates DNMT1, leading to hypomethylation of DNA. From the increased protein expression of tumor-suppressor genes and functional activation of PCFT, we concluded that RX-3117 might have induced hypomethylation of the promotor.

Additional file 2: Table S1. Antibodies used in these studies. Antibody Sources, Clone ID and Fluorochromes are as indicated.

Additional file 1: Figure S1. No difference observed in frequency of T regulatory cells (CD25 + FoxP3+) among human CD4+ T cells in the tumors of control or treated mice. A) Representative flow plots illustrating detection of Tregs by CD25 and FoxP3 and B) frequency of Tregs among CD4+ T cells in the lymph nodes (LN) and tumors (TIL) for each mouse, according to treatment. Bars represent arithmetic means.

Background: RX-5902 (Supinoxin) is a novel anti-cancer compound that targets phosphorylated p68 RNA helicase, a member of the DEAD box family of helicases, affecting upstream and downstream molecules in the Wnt canonical pathway. As a single agent, RX-5902 exhibits strong growth inhibition in both in vitro and in vivo settings. Specifically, RX-5902 enhances survival and tumor growth inhibition in numerous xenograft models, including ovarian, renal and breast. We have previously shown RX-5902 inhibits cell growth in a dose-dependent fashion in the triple-negative breast cancer (TNBC) xenograft MDA-MB231. In the current study, we have expanded our investigation of the therapeutic potential of RX-5902 against TNBC using both in vitro and in vivo preclinical models. Methods: RX-5902 was provided by Rexahn, Inc. (Rockville, MD). Cell proliferation was measured using the Cell-Titer Glo luminescent cell viability assay (Promega). Apoptosis was assessed using Incucyte Caspase 3/7 Green apoptosis assay (Essenbioscience). Immunoblots of MDA-MB-231 cell line were probed for ß-catenin (Cell Signaling). Syngeneic 4T1 murine TNBC mice were obtained from Sippr-BK Laboratory Animal Co (Shanghai, China) and tumor volumes were measured twice a week. When the mean tumor volumes reached ˜90 mm3, mice were randomized and treated with vehicle or RX-5902 PO daily alone or in combination with anti-CTLA4 or anti-PD-1 BIW for 3 weeks. Tumor growth inhibition (TGI) was calculated at Day 25. Results: A panel of 18 TNBC cell lines were treated with RX-5902 and effects on cell proliferation were measured by the Cell Titer-Glo assay. Using 100nM as a cutoff, 14 sensitive lines and 4 resistant lines were identified, with an average IC50 of 56 nM in the sensitive lines. Of these, we chose 2 sensitive lines (MDA-MB-231, HCC1806) and 2 resistant lines (MDA-MB-436 and CAL-120) and assessed induction of apoptosis by the Incucyte caspase activity assay. Robust induction of apoptosis was observed in both sensitive lines (N=3). These lines were then subjected to cell cycle analysis by flow cytometry, which revealed a pronounced G2/M cell cycle arrest and aneuploidy following exposure to RX-5902. Western blot analysis of the MDA-MB-231 cell line showed decreases in the Wnt pathway-related protein nuclear ß-catenin in doses ranging from 20 nM to 200 nM. Finally, the therapeutic efficacy of RX-5902 was assessed as a single agent and in combination with two immune-oncology agents in the treatment of the TNBC 4T1 animal model. RX-5902 as a single agent showed dose dependency in the 4T1 model, and when given in combination with either anti-CTLA4 or anti-PD1 showed an additive effect (p Conclusions: RX-5902 showed efficacy against several in vitro and in vivo preclinical models of TNBC. RX-5902 resulted in G2/M arrest and induced apoptosis in sensitive TNBC cell lines and decreases in nuclear beta-catenin. In vivo, RX-5902 demonstrated additive anti-tumor effects when combined with either anti-CTLA4 or anti-PD1 immunotherapies. Together, these finding indicate that RX-5902 may have important clinical implications for the treatment of TNBC. A phase 2a clinical study in metastatic TNBC is ongoing..training_cert Citation Format: Tentler JJ, Frank JG, Kim DJ, George C, Lee YB, Ely B, Tan AC, Kim J, Pitts TM, Capasso A, Dailey KL, Eckhardt G, Diamond JR. Preclinical studies of RX-5902, a beta-catenin modulator in triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-16.

RX-0201 is an antisense oligonucleotide (ASO) against Akt1 currently in clinical trial for metastatic renal cancer.To improve the delivery of RX-0201 using folate receptor-targeted lipid-albumin nanoparticles (F-LAN).F-LAN were synthesized with the composition of DOTAP/soyPC/TPGS/folate-PEG-DSPE (25:70:4:1 m/m), a cationic human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate and RX-0201. The nanoparticles were evaluated in KB human carcinoma cells in vitro and in a KB murine xenograft tumour model in vivo for pharmacokinetics and antitumor activities.The F-LAN-RX-0201 had a mean particle size of 108.6 ± 5.8 nm, zeta potential of 10.5 ± 3.2 mV and ASO loading efficiency of 71.5 ± 4.5%. In KB cells, uptake and Akt1 inhibition by F-LAN-RX-0201 were greater than those of non-targeted LAN-RX-0201 and could be partially blocked by excess free folate. F-LAN-RX-0201 inhibited cell growth with an ICF-LAN-RX-0201 showed promise as a therapeutic agent for tumours with elevated folate-receptor expression.

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of β-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on β-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2–M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear β-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFβ, and Wnt/β-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear β-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

Lipid-albumin nanoparticles (LAN) were synthesized for delivery of RX-0047, an antisense oligonucleotide (ASO) against the hypoxia inducible factor-1 alpha (HIF-1α) to solid tumor. These lipid nanoparticles (LNs) incorporated a human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, which is cationic and can form electrostatic complexes with negatively charged oligonucleotides. The delivery efficiency of LAN-RX-0047 was investigated in KB cells and a KB murine xenograft model. When KB cells were treated with LAN-RX-0047, significant HIF-1α downregulation and enhanced cellular uptake were observed compared to LN-RX-0047. LN-RX-0047 and LAN-RX-0047 showed similar cytotoxicity against KB cells with IC50 values of 19.3 ± 3.8 and 20.1 ± 4.2 μM, respectively. LAN-RX-0047 was shown to be taken up by the cells via the macropinocytosis and caveolae-mediated endocytosis pathways while LN-RX-0047 was taken up by cells via caveolae-mediated endocytosis. In the KB xenograft tumor model, LAN-RX-0047 exhibited tumor suppressive activity and significantly reduced intratumoral HIF-1α expression compared to LN-RX-0047. Furthermore, LAN-RX-0047 greatly increased survival time of mice bearing KB-1 xenograft tumors at doses of either 3 mg/kg or 16 mg/kg. These results indicated that LAN-RX-0047 is a highly effective vehicle for therapeutic delivery of antisense agents to tumor.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of 3H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the β-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the β-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - β-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound. J. Cell. Biochem. 116: 1595–1601, 2015. © 2015 Wiley Periodicals, Inc.

RX-3117 is a novel cytidine analog showing encouraging results in ongoing Phase 2a studies with gemcitabine-resistant pancreatic ductal adenocarcinoma and advanced urothelial bladder cancer. Cellular uptake of RX-3117 is mediated by the equilibrative nucleoside transporter 1 (hENT1). RX-3117 is activated by uridine-cytidine kinase-2 (UCK2) and is not a substrate for cytidine deaminase (CDA), and shows a good oral bioavailability. In an earlier study accumulation of RX-3117 nucleotides was associated with sensitivity to the drug. RX-3117 is incorporated into RNA and DNA and inhibits DNA methyltransferase 1 (DNMT1). RX-3117 is active against gemcitabine-resistant non-small cell lung cancer (NSCLC) cell lines. In this study we aimed to elucidate potential resistance mechanisms against RX-3117. For that purpose the NSCLC cell lines H460, A549 and SW1573 (including its gemcitabine resistant variant SW-G) were exposed to increasing concentrations of RX-3117. For A549 and SW1573 two variants were obtained: A549/RX1, A549/RX2, SW1573/RX1 and SW1573/RX2. Resistance varied from 10 (H460; SW-G) to >100-fold for the other cell lines. Resistance was stable except for H460 cells. Cross-resistance in the same range was observed for the cytidine analogs ethynyl-cytidine (ETC), cyclopentenylcytosine (CPEC) and aza-cytidine (aza-CR), which are all activated by UCK2; no cross-resistance was observed for gemcitabine and aza-deoxycytidine which are activated by deoxycytidine kinase. No change was observed in the expression of DNMT1, while expression of ribonucleotide reductase 1 was decreased. However, analysis of UCK2 at the enzyme activity level, protein and gene expression did not reveal a decrease in UCK2 while for ETC, aza-CR and CPEC resistance was reported to be mediated by a UCK2 deficiency. No change was found in the other activating enzymes UMP-kinase, CMP-kinase and NME1/NDKA. Despite this lack of change, accumulation of RX-3117 and RX-3117 nucleotides (mono-, di- and triphosphate) was 2-5-fold lower in the resistant cells after incubation with 10 or 100 μM RX-3117 for 4-24 hr. No evidence for accumulation of RX-3117 deoxynucleotides was found in both the parent and the resistant cell lines. Since no change in hENT1 was found, cell lines were further investigated by using RNA-seq, and western blotting focusing on nucleoside/nucleotide degradation enzymes. CDA was decreased in resistant cells, but the cytosolic pyrimidine nucleotidase NT5C3 was increased in the resistant cell lines as well as DCTPP1, which can degrade deoxynucleoside triphosphates. Moreover several DNA repair enzymes SAMHD1, MTH1 and TDP1 were increased. However, inhibition of MTH1 and DCTPP1 did not increase sensitivity to RX-3117. In conclusion, induction of resistance to the novel cytidine analog RX-3117 led to a decreased accumulation of RX-3117 ribonucleotides, which might be associated with an increased degradation. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Btissame El Hassouni, Richard J. Honeywell, Joris R. Julsing, Beatrice Balboni, Daniel J. De Klerk, Safet Zekanovic, Kees Smid, Elisa Giovannetti, Young B. Lee, Deog J. Kim. Unusual mechanism of resistance to the novel cytidine analog fluorocyclopentenylcytosine (RX-3117) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1267.

Phosphorylated p68 (p-p68 or phospho-DDX5) has been shown to be associated with cell transformation, epithelial mesenchymal transition (EMT) and cell migration, and furthermore play a vital role in cell proliferation and tumor/cancer progression by promoting the nuclear translocation of β-catenin. RX-5902 is an oral, small molecule inhibitor of β-catenin nuclear translocation mediated by p-p68. Previous studies have shown that RX-5902 inhibits the growth of cancer cells at low nanomolar IC50 (10 to 24nM) and disrupts the p-p68-β-catenin signaling pathway through an interaction with p-p68 on Tyr 593. By interfering with the p-p68-β-catenin signaling pathway cancer cells undergo apoptosis. In this study, we expanded our investigation to study the effect of RX-5902 on nuclear β-catenin in several types of cancer cell lines that include colon, gastric, hepatic, head and neck, melanoma, breast, pancreatic, ovarian, and prostate. Western blot data, in the cell lines tested, showed a decrease of nuclear β-catenin protein as well as p-p68 and c-myc in a concentration-dependent manner. Furthermore, an in vivo study of the MDA-MD-231 xenograft model demonstrated the ability of RX-5902 to inhibit the tumor growth by decreasing p-p68, β-catenin, cyclin D1 and c-myc proteins. Collectively our findings in both in vitro and in vivo studies support the potential therapeutic effect of RX-5902 in multiple cancer indications through the disruption of the phospho-p68/nuclear β-catenin interaction and blocking the nuclear translocation of β-catenin. Citation Format: Young B. Lee, Christina George, Deog J. Kim. The anticancer effects of RX-5902 result from inhibition of phosphorylated p68-mediated β-catenin nuclear translocation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1298.

Background: RX-3117 is an oral, small molecule nucleoside analogue pro-drug being used in combination with nab-paclitaxel in a Ph2 clinical trial for pancreatic cancer. RX-3117 can be transported intracellularly by SLC29A1 (hENT1) and is converted to an active agent by the cancer-enriched enzyme, uridine-cytidine kinase 2 (UCK2) for incorporation into RNA and DNA. This leads to cancer cell apoptosis. We developed an approach to enumerate and phenotype CTCs from pancreatic cancer subjects by quantitative immunofluorescence (QIF) to assess whether CTC numbers, phenotypic features and/or early apoptotic responses to therapy predict or presage clinical response. Methods: QIF staining parameters were developed with cancer cell lines sensitive and resistant to RX-3117 utilizing monoclonal antibodies to hENT1 and UCK2. The staining methods were then applied to CTCs isolated from 10 ml anticoagulated blood by dielectrophoretic (DEP) properties that differentiate cancer cells and normal PBMCs using the ApoStream device (ApoCell, TX). In addition to the drug-related markers, CTCs were stained for a panel of either Epithelial or Mesenchymal markers, CD45 and nuclei (DAPI). Phenotyped CTCs were binned into 6 categories defined by whether they were EPI+/-, EMT+/- or CD45+/-. The percentage of cells in each category also positive for hENT1 or UCK2 was determined as was the mean fluorescence intensity of the marker+ sub-population. In addition, assessment of hENT1 and UCK2 expression was performed by qRT-PCR utilizing mRNA isolated from isolated CTCs, the buffy coat from spun blood and in plasma. Results: In a pilot study, blood was obtained from five subjects with Stage IV pancreatic cancer on various regimens. CTCs were isolated, enumerated and phenotyped. CTC numbers ranged from 100 – 20,000 per ml. The percentages of UCK2+ cells were higher in EPI+/EMT+ subsets than EPI-/EMT- subsets. The percentages of hENT1+ cells were generally low in all the subsets. hENT1 and UCK2 transcripts were observed at acceptable levels in the cell populations, and borderline, but consistent levels in plasma samples. CEACAM5 transcripts were observed only in one CTC and PBMC fraction from one subject. After normalization vs. housekeeping transcripts, a greater than 2-fold increase of UCK2 was observed in three CTC samples compared to PBMC samples. A greater than 2-fold increase of hENT1 was observed in two CTC samples. Conclusion: CTCs from patients with pancreatic cancer can be obtained in numbers suitable for multiparameter phenotyping to identify features that might serve as selective, predictive or prognostic biomarkers in clinical trials. Multiparameter phenotyping of several relevant markers is being performed by quantitative immunofluorescence on CTCs isolated from pancreatic cancer patients in an on-going Ph2 clinical trial of RX-3117 in combination with nab-paclitaxel. Citation Format: Andrew Eisen, DJ Kim, Jie Yang, Weiguo Wu, Darren W. Davis. Phenotyping pancreatic cancer CTCs as biomarkers for RX-3117 clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 428.

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90 % inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.

RX-3117 (fluorocyclopentenyl-cytosine) is a novel cytidine analog currently being evaluated in a Phase Ib clinical trial in cancer patients with solid tumors. The radiosensitizing effect of RX-3117 was studied in A2780 ovarian cancer cells and non-small cell lung cancer cell lines and related to cell survival and the effect on cell cycle and cell cycle proteins. RX-3117 has a schedule-dependent radiosensitizing effect, but only at pre-incubation (dose modifying factors: 1.4–1.8), observed at pulse and fractionated irradiation. Radiosensitizion was also seen in a three-dimensional spheroid model. At the low radiosensitizing concentration, RX-3117 in combination with radiation led to an accumulation of cells in S-phase, which was accompanied with an increase of cell cycle proteins such as p-Chk2 and p-cdc25C. In addition, RX-3117 caused DNA damage and increased apoptosis. In conclusion, our in vitro experiments showed a radiosensitizing effect of RX-3117.

Clavulanic acid is a psychoactive compound that has been shown to modulate central nervous system activity. Importantly, in neurotoxin-induced animal models, clavulanic acid has been shown to improve motor function (Huh et al., 2010) suggesting that it can be neuroprotective; however, the mechanism as how clavulanic acid can induce neuroprotection is not known. We demonstrate here that clavulanic acid abrogates the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) which mimics Parkinson's disease (PD) by inducing neurodegeneration. To further establish the mechanism we identified that clavulanic acid inhibits neurotoxin-induced loss of mitochondrial membrane potential and ROS production. Consistent with these results, neurotoxin-induced increase in Bax levels was also decreased in clavulanic acid treated cells. Importantly, neurotoxin-induced release of cytochrome c levels as well as caspase activation was also inhibited in clavulanic acid treated cells. In addition, Bcl-xl levels were also restored and the Bcl-xl/Bax ratio that is critical for inducing apoptosis was increased in clavulanic acid treated cells. Overall, these results suggest that clavulanic acid is intimately involved in inhibiting neurotoxin-induced loss of mitochondrial function and induction of apoptosis that contributes towards neuronal survival.

Human norepinephrine (NE) deficiency (or dopamine β-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).

RX-3117 is a novel, investigational, oral, small molecule nucleoside compound that shows promising antitumor activity in xenografts including patient-derived xenografts resistant to gemcitabine. RX-3117 is currently being evaluated in a Phase IIa multi-center, open-label clinical study in patients with relapsed or refractory pancreatic cancer and advanced bladder cancer. RX-3117 can downregulate DNMT1 and activate genes controlled by methylation, such as E-cadherin and p16. We aimed to elucidate the mechanism of RX-3117 mediated downregulation of DNMT1. Expression of DNMT1 was evaluated by western blotting. Binding of DNMT1 to DNA was measured after DNA isolation, DNAse treatment and gel electrophoresis. DNMT1 trapping in the nucleus was also studied with ImageStream FACS analysis. DNMT1 ubiquitination was measured by immunoprecipitation and quantification of the ubiquitin tag K48. Translocation of DNMT1 was studied by confocal microscopy. Modeling studies were performed with Quantum Mechanics (QM) and Molecular Dynamics (MD). DNMT1 acts on the DNA during the so-called base flipping necessary for methyl transfer. To determine whether RX-3117 treatment would lead to trapping of DNMT1, NSCLC A549 and PDAC SUIT-028 cells were treated for 24 hr with 1 µM RX-3117. Immunostaining of DNMT1-DNA and ImageStream FACS analysis demonstrated binding of DNMT1 to DNA. Treatment with RX-3117 also resulted in DNMT1 ubiquitination, as shown by an increase in the ubiquitin tag K48, reaching the highest level after 48 hr. Incubation of A549 and SUIT-028 cells with 80 nM of the specific proteasome inhibitor bortezomib prevented RX-3117 mediated degradation of DNMT1 (at 24 and 24 hr exposure to 1 µM RX-3117). This underlines the role of the proteasome in DNMT1 degradation. With confocal microscopy it was demonstrated that DNMT1 and the proteasome co-localized and translocated to the cytosol of A549 and SUIT-028 cells treated for 48 hours with 1 µM RX-3117. With molecular modelling using QM and MD, changes in nucleophilicity of C4-C5 double bound were observed indicating that DNMT1 would initiate methyl transfer but cannot proceed. In summary we conclude that RX-3117 induces epigenetic changes in cancer cells by trapping DNMT1 in the nucleus followed by translocation of the protein to the proteasome for degradation. Citation Format: Dzjemma Sarkisjan, Ferry Pronk, Rosan Kuin, Btissame el Hassouni, Young B. Lee, Deog J. Kim, Marc Dijk, Daan P. Geerke, G. J. Peters. RX-3117 promotes epigenetic effects in cancer cells through enhanced degradation of DNMT1 [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 5861.

Clavulanic acid is a psychoactive compound with excellent blood-brain barrier permeability and safety profiles. Previous studies showed that clavulanic acid suppresses anxiety in rodents and in a primate model. In addition, clavulanic acid is thought to enhance sexual function in animal models via central nervous system (CNS) mechanisms. To further examine its potential as a CNS-modulating agent, we investigated the effects of clavulanic acid in neurotoxin-induced animal models that emulate neurodegenerative disease symptoms. Clavulanic acid was administered to rodents that were exposed to kainic acid or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using histochemical staining of brain sections, we demonstrated that clavulanic acid protects hippocampal and dopaminergic neurons from toxin-induced acute death. We also observed that clavulanic acid improves motor function in MPTP-treated mice in a behavioral test. These data indicate that clavulanic acid may have neuroprotective effects and warrants further investigation of its therapeutic use in CNS disorders, such as Parkinson's and Alzheimer's disease. Drug Dev Res 71:351–357, 2010. © 2010 Wiley-Liss, Inc.

Sexual behaviour in rats can be used to predict putative effects on human sexual behaviour. Anecdotic reports exist, that the beta-lactamase inhibitor, clavulanic acid exerts sexual stimulating activities in monkeys. To characterize these pro-sexual activities, clavulanic acid was tested in three doses and compared to one dose of a sexually inhibitory dose of the selective serotonin reuptake inhibitor, paroxetine, in sexually-experienced male rats, selected for a moderate level of sexual performance in a standard 30-min test with an oestrus female. After acute administration, clavulanic acid had minor sexual stimulating effects at the highest dose in the number of intromissions and in the first ejaculation series. After sub-chronic 7-days treatment, clavulanic acid increased the number of ejaculations at all three doses and reduced the number of intromissions in the 1st series at the highest dose. After chronic 14 days treatment, a similar but stronger pro-sexual profile was observed. The sexual side effects of paroxetine were as expected, including slight sexual inhibitory effects after acute administration, but somewhat stronger overall inhibitory effects after 7 and 14-days pretreatment, particularly notable in the decreasing number of animals contributing to the 2nd ejaculation series, which was even stronger after 14-days treatment. One week after cessation of treatment, the paroxetine group had completely recovered, whereas the highest dose-group of clavulanic acid still showed some pro-sexual effects. This remarkable pro-sexual activity of clavulanic acid cannot readily be explained by its mechanism of action as a beta-lactamase inhibitor but could be due to unexpected central activity of the compound.

RX-3117 (fluorocyclopentenylcytosine) is a cytidine analog and this class of drugs, including gemcitabine, has been widely used for the treatment of various types of cancers. However, there is no oral formulation of gemcitabine and drug resistance to gemcitabine is common. In this study, the efficacy of orally-administered RX-3117 was examined in 9 different human tumor xenograft models (colon, non-small cell lung, small cell lung, pancreatic, renal and cervical), grown subcutaneously in athymic nude mice. In the Colo 205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 30%, 25% and 0% tumor growth inhibition (TGI), respectively, whereas oral treatment with RX-3117 induced 100%, 78%, 62% and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. RX-3117 was also evaluated in a single primary low-passage human pancreatic Tumorgraft™CTG-0298 (TGI 76%), which is relatively resistant to gemcitabine (TGI 38%) and has a favorable RX-3117-activating enzyme profile. These studies demonstrated the therapeutic potential and anticancer efficacy of RX-3117.

Background: A novel, orally bioavailable nucleoside analogue, RX-3117, is a prodrug activated intracellularly by Uridine Cytidine Kinase 2 (UCK2) that is thought to be expressed predominantly in tumor tissue. RX-3117 is currently being evaluated in a Phase Ib/IIa multi-center, open-label clinical study in patients with advanced pancreatic and bladder cancer. In this study, we aimed to determine the relation between UCK2 tissue protein expression and the efficacy of RX-3117 in mice xenograft models and also UCK2 protein expression in a panel of human cancer tissues relative to normal tissue. Methods: The UCK2 protein expression in tumor tissues was analyzed by immunoblotting using clone 22-1 rabbit monoclonal antibody. The validated procedure for the immunohistochemistry (IHC) of UCK2 with clone 22-1 was performed in a panel of human formalin-fixed paraffin-embedded (FFPE) cancer and normal tissues. Results: The immunoblotting protein level of UCK2 normalized to β-actin and corresponding tumor growth inhibition (oral RX-3117 dose of 500mg/kg, TIWK) were 57 and 67% in MiaPaCa2, 30 and -5% in BxPC3, 199 and 92% in Colo-205, 21 and 90% in Caki-1, 2 and 39% in A549, and 146 and 79% in H460, respectively. This data indicates an anti-tumor efficacy trend in a UCK2-dependent manner. The IHC of UCK2 showed that positive staining of UCK2 in cancer tissues was observed in 20/20 bladder cancer tissues (100% frequency), 19/20 CRC tissues (95% frequency), 18/20 NSCLC tissues (90% frequency), and 19/20 pancreatic cancer tissues (95% frequency). Average H-Scores of UCK2 in cancer tissues vs. normal tissues were 104 vs. 9 in lung, 97 vs. 20 in bladder, 67 vs. 41 in pancreas and 39 vs. 21 in colon, respectively. Conclusions: The current data showed a correlation trend between UCK2 protein expression level and degree of antitumor activity of RX-3117 in xenograft models. It also supports a higher UCK2 protein expression level in human cancer tissues compared to their normal tissues. This suggests that RX-3117 activity may be specific to tumor tissue, and quantification of UCK2 expression in human cancer tissues may be useful as a predictive biomarker to select patients for their sensitivity to RX-3117 in future clinical studies. Citation Format: Young Bok B. Lee, Deog J. Kim, Christina George, Chang-ho Ahn, Julie Frank, Reza Mazhari, Lisa M. Dauffenbach, Eric P. Olsen. Evaluation of UCK2 protein expression as a potential predictive biomarker of RX-3117 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1790. doi:10.1158/1538-7445.AM2017-1790

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of (3) H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the β-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the β-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - β-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound.

SummaryMouse factor X was highly purified from plasma using barium ion precipitation and chromatography on anion-exchange and heparin- agarose affinity chromatography columns. Intact and reduced patterns of mouse factor X in SDS-PAGE were similar to those of human factor X. The specific absorption E 1%/1 cm at 280 nm of mouse factor X was found to be 11.2. Content of carbohydrate moieties of mouse factor X, determined to be 10% by weight, differs both quantitatively and quantitatively from that of human factor X, while the γ-carboxy- glutamic acid (Gla) and β-hydroxy-aspartic acid (β-OH-Asp) content were essentially the same as for human factor X. The amino-terminal amino acid sequences of the light and heavy chains of mouse factor X separated by SDS-PAGE were ANSFF--FKK and SVALXTSDSE, respectively. Underlined residues are non-identical with those of human factor X. Clotting time-based assays using human factor X- deficient plasma as substrate exhibited the following apparent extents of activation of factor X in mouse plasma, using human plasma as the standard: 195% (intrinsic); 200% (extrinsic); and 190% (RVV-X). Using the purified proteins in the same assay systems, the following apparent activation of mouse factor X was demonstrated, compared with human factor X: 195% (intrinsic); 27% (extrinsic); and 41% (RVV-X). These activity profiles suggest that the human extrinsic coagulation pathway functions less efficiently than the corresponding mouse pathway in the activation of mouse factor X. Furthermore, mouse brain thromboplastin satisfactorily replaced rabbit brain thromboplastin in extrinsic activation of factor X in mouse plasma, but not of human plasma or purified mouse or human factor X, in line with studies by others suggesting that human factor Vila poorly activates factor X in the presence of mouse tissue factor. While fully RVV-X-activated mouse factor X activated human prothrombin at a rate equal to about 117% of that for human factor X, it hydrolyzed the synthetic substrate, S-2222, at a rate of only about 18% of that for human factor X. These results are expected to be useful in making the mouse suitable for study of the mammalian blood coagulation pathways.

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10–20% of normal) and antigen (30–35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13 984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors XWenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

Akt-1 is a protein kinase that contributes to cancer progression by promoting proliferation and inhibiting apoptosis of tumor cells. RX-0201 (Archexin®), a 20-mer phosphorothioate antisense oligonucleotide that inhibits the expression of Akt-1, is currently in a Phase IIa clinical trial for metastatic renal cancer. To further improve the efficacy of RX-0201, folate receptor-targeted lipid coated albumin nanoparticles containing RX-0201 (F-LCAN-RX-0201) were synthesized and evaluated for Akt-1 inhibition and antitumor activity. These nanoparticles are composed of DOTAP/soyPC/TPGS/folate-PEG-DSPE (25:70:4:1), a cationic human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, and RX-0201. F-LCAN-RX-0201 had a particle size of 108.6±5.8 nm, zeta potential of 10.5±3.2 mV and 71.5% antisense loading efficiency. F-LCAN-RX-0201 in folate receptor-expressing KB cells inhibited cell growth at IC50 of 11.9 ± 4.0 μM while free RX-0201 did not show significant cytotoxicity at concentration up to 250 μM. Cellular uptake and Akt-1 inhibition of F-LCAN-RX-0201 was greater than the non-targeted LCAN-RX-0201 and was partially blocked by excess free folate. Pharmacokinetic studies showed that F-LCAN-RX-0201 had a longer half-life than that of free RX-0201 (30 ± 4 min vs 10 ± 2 min). In a KB xenograft tumor model, F-LCAN-RX-0201 exhibited higher tumor suppressive activity compared to the non-targeted LCAN-RX-0201 at 16 mg/kg (as a RX-0201 amount) without body weight changes. Moreover, the antitumor activity of F-LCAN-RX-0201 at a dose of 16mg/kg (as a RX-0201 amount) was similar to free RX-0201 at a dose of 90mg/kg. In conclusion, LCAN is a highly effective formulation for therapeutic delivery of antisense agents and F-LCAN-RX-0201 could be a potential therapeutic agent for tumors with increased folate-receptor expression. Citation Format: Hong Li, Xinwei Cheng, Yang Liu, Young Bok Lee, Deog Joong Kim, Chang-ho Ahn, Robert J. Lee. Folate receptor-targeted lipid coated albumin nanoparticles (F-LCAN) for therapeutic delivery of RX-0201 (Archexin®), an antisense oligonucleotide against Akt-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1322.

RX-3117 (Fluorocyclopentenylcytosine; FCPEC) is an orally bioavailable novel cytidine analog which is currently being evaluated in a Phase I clinical study. Downregulation of DNA methyltransferase 1 (DNMT1) by RX-3117 was shown in various cell lines with different histological backgrounds. In the present study we determined the effect of RX-3117 on DNMT1 at the DNA, RNA, protein and enzyme activity, and reactivation of suppressed target genes, including p16INK4A, methylguanine methyltransferase (MGMT) and the proton coupled folate transporter (PCFT). In the moderately sensitive non-small cell lung cancer (NSCLC) cell lines such as A549 and SW1573, 5-50 μM RX-3117 downregulated DNMT1 protein expression by 5-20% after 24 hr exposure and >90% after 48 hr. DNMT1 mRNA was not affected after 24 hr exposure but was affected moderately after 48 hr. In the sensitive ovarian cancer cell line A2780, protein down regulation was already observed after 24 hr at 1 μM RX-3117. DNMT1 activity was inhibited by 1 μM RX-3117 by 32% which was similar to the percent inhibition with 5 μM of the reference compound 5-aza-2’-deoxycytidine (DAC, 31%). In A549 cells 5 μM RX-3117 decreased overall methylation of DNA (detected by an antibody against 5-methylcytosine) by 25% after 48 hr exposure, while 5 μM DAC only inhibited 9%. For several genes known to be affected by methylation, we evaluated their protein expression and activity. In A549 and SW1573 cells a 24 hr exposure to 5 μM RX-3117 increased the expression of the cell cycle protein p16INK4A and of the DNA repair enzyme MGMT. For PCFT we evaluated its functional activity in CCRF-CEM leukemic cells which have a highly methylated PCFT promoter and in CEM-MTX cells which are deficient for the reduced folate carrier (RFC). PCFT is a specific folate transporter responsible for uptake of folic acid and the folate analogs methotrexate (MTX) and pemetrexed. Incubation of both CEM and CEM-MTX cells with either 29.6 μM RX-3117 or DAC as a positive control markedly increased PCFT mediated transport of MTX. This was more pronounced in CEM-MTX cells, 10-11-fold increase for both RX-3117 and DAC, compared to a 4-fold increase in CEM cells. In conclusion, the down-regulation of DNMT1 by RX-3117 was accompanied by a decrease in DNMT1 protein expression and enzyme activity, resulting in up-regulation of proteins of MGMT, PCFT, and the tumor suppressor gene p16INK4A. These data underline DNMT1 inhibition as a novel mechanism of RX-3117. Citation Format: Godefridus J. Peters, Dzjemma Sarkisjan, Joris J. Julsing, Ahmed Hassan, Kees Smid, Ietje Kathmann, Young Lee, Deog J. Kim. Inhibition of DNA methyltransferase by RX-3117 (fluorocyclopentenylcytosine) leads to upregulation of hypomethylated targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4725.

Several studies have indicated that the DEAD box RNA helicase DDX5/p68 plays several important roles in cancer (1, 2). In particular, p68 that is phosphorylated on Tyr593 has been shown to be associated with cell transformaton, epithelial mesenchymal transition (EMT) and cell migration (3). Therefore, phosphorylated p68 may be a promising target for novel anti-cancer therapeutics. We previously reported that 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl) aminocarbonyl] piperazine (RX-5902, Supinoxin®) inhibits the growth of cancer cells at low nanomolar concentrations by interacting with phosphorylated p68 on Tyr593, interfering with the phosphorylated p68-β-catenin signaling pathway (4). In this study, we sought to determine whether phosphorylated p68 on Tyr593 plays a key role in RX-5902's ability to inhibit cancer cell growth by knocking down p68. p68-siRNA efficiently down-regulated the expression of phosphorylated p68 on Tyr593 as well as p68 in the triple-negative (TN) breast cancer cell line, MDA-MB-231. Exposure of p68-siRNA-transfected cells to the IC50 concentration of RX-5902 protected MDA-MB-231 cells from the cytotoxic effects of RX-5902, indicating the phosphorylated p68 on Tyr593 is a key molecule for RX-5902 cytotoxic effects. We also examined the tumor growth inhibition (TGI) of RX-5902 in the human TN-breast tumor (MDA-MB-231) xenograft mouse model. Not only did RX-5902 demonstrate potent efficacy in this model but also oral administration with RX-5902 resulted in dose-dependent TGI and extended the overall survival of these animals. Oral administration of 160, 320 and 600 mg/kg of RX-5902 showed 54.4%, 84.4% and 100% TGI, respectively whereas 5 mg/kg of Abraxane (iv) showed only 48.2% TGI at day 29. Further studies demonstrated the inhibitory effects of RX-5902 on cellular motility in MDA-MB-231 in wound healing assays, suggesting the potential function of phosphorylated p68 on Tyr593 in cell migration (5). These data support the potential therapeutic activity of RX-5902 in triple negative breast cancers. A Phase 1 study of RX-5902 on relapse/refractory solid tumors is ongoing. References 1. Fuller-Pace, FV, RNA Biology 10, 121–132 (2013) 2. Dai et al. Journal of Experimental & Clinical Cancer Research, 33, 64-71 (2014 3. Yang et al., Cell, 127, 139–155 (2006) 4. Kost et al., Journal of Cellular Biochemistry ;online: 3 FEB 2015 05:14PM EST | DOI: 10.1002/jcb.25113) (2015). 5. Remenyi et al, presented at 2015 AACR (2015). Citation Format: Kim DJ, Yang MY, Lee YB, Remenyi J, Fuller-Pace F. The anticancer effects of Supinoxin® (RX-5902) in triple-negative breast cancer MDA-MB-231 through phosphorylated p68 on Tyr593. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-13.

238 Background: DEAD box RNA helicase DDX5/p68, and its phosphorylated form in particular, may play several important roles in cancer by means of cell transformation, epithelial mesenchymal transition (EMT), and cell migration, deeming it a promising target for novel anti-cancer therapeutics. We have previously shown that Supinoxin (RX-5902) interacts with phosphorylated p68 on Tyr593, interfering with the phosphorylated p68-β-catenin signaling pathway. Supinoxin also inhibits motility of MDA-MB231 breast cancer cell lines and could potentially prevent metastasis in cancer. Methods: In this study, we first demonstrated anti-proliferative effects in several cell lines originated from pancreas (5 cell lines) with a high level of sensitivity to Supinoxin (IC50 of 18 nM). We also sought to examine the tumor growth inhibition (TGI) and/or tumor growth delay (TGD), and survival benefits of Supinoxin in the human pancreatic carcinoma (MiaPaCa-2) xenograft mouse model. Results: In the MiaPaCa-2 model, at 50 and 70 mg/kg daily (oral, 5 days on/2 days off) for 3 weeks, both doses delayed tumor growth significantly (83% and 339%; P < 0.001). All animals in the high dose showed complete regression (CR) and completing the study as tumor free survival (TFS; Day 65); one animal showed CR and TFS in the lower dose. Gemcitabine (120 mg/kg ip; Q3D for 4 weeks) resulted in TGD of 71% (P < 0.001), with no CRs. Supinoxin did not result in a reduction in body weight gain, treatment related deaths, or clinical observations in this tumor model. Conclusions: These data support the potential therapeutic activity of Supinoxin in pancreatic cancer. A Phase 1 study of Supinoxin on relapse/refractory solid tumors is ongoing (NCT02003092).

524 Background: DEAD box RNA helicase DDX5/p68 may play several important roles in cancer. In particular, phosphorylated p68 has been shown to be associated with cell transformation, epithelial mesenchymal transition (EMT), and cell migration, deeming it a promising target for novel anti-cancer therapeutics. We have previously shown that Supinoxin (RX-5902) interacts with phosphorylated p68 on Tyr593, interfering with the phosphorylated p68-β-catenin signaling pathway. Methods: In this study, we first demonstrated anti-proliferative effects in ten renal cancer cell lines with a high level of sensitivity to Supinoxin (IC50 of 39 nM); TK-10 was the only resistant cell line in this study. We also sought to examine the tumor growth inhibition (TGI), tumor growth delay (TGD), and survival benefits of Supinoxin in the human renal cell carcinoma tumor (Caki-1) xenograft mouse model, using two different dosing schemas: weekly dosing at 20-160 mg/kg for 4 weeks, or 50-70 mg/kg daily (5 days on/2 days off) for 3 weeks. Results: Weekly dosing of Supinoxin at 160 mg/kg resulted in a 75% TGD (P < 0.001). Daily administration of Supinoxin resulted in dose-dependent TGI (80 and 96%; Day 21) and TGD (68 and 104%, P < 0.001), and extended the overall survival of the animals at both doses (P < 0.0001). At the dose of 70 mg/kg daily, 6/10 animals demonstrated partial tumor regressions and 1/10 a complete tumor regressions. Supinoxin did not result in a reduction in body weight gain, treatment related deaths, or clinical observations in either of the dosing schemas. Sunitinib (60 mg/kg; daily for 21 days) resulted in TGD for both in vivo studies validating the Caki-1 model herein. Conclusions: These data support the potential therapeutic activity of Supinoxin in renal cell cancers. A Phase I study of Supinoxin on relapse/refractory solid tumors is ongoing (NCT02003092).

Human embryonic kidney cells (293) were transfected with a construct containing human factor X cDNA and selected for G418 resistance. The level of expression of recombinant factor X in serum-free medium was 4 to 5 μg/ml. Purified recombinant factor X had a molecular size identical to that of normal plasma factor X. Amino-terminal sequencing revealed normal processing cleavages. The γ-carboxy Glu and β-OH Asp content of the recombinant factor X was close to 90% of the expected levels of these post-translational residues. The specific activity of recombinant factor X was about 95% of that of plasma factor X in three plasma-based clotting assays. This report demonstrates that 293 cells can produce a high level of biologically active factor X and describes a visual criterion for verifying the transfection process.

Based on the anticancer activity of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives published in Bioorg. Med. Chem. 2010 , 18 , 7966, we further explored the synthesis of 7 or 8-substituted quinoxalinyl piperazine derivatives. From in vitro studies of the newly synthesized compounds using human cancer cell lines, we identified some of the 8-substituted compounds, for example 6p , 6q and 6r , which inhibited the proliferation of various human cancer cells at nanomolar concentrations. Compound 6r , in particular, showed the lowest IC 50 values, ranging from 6.1 to 17 nM, in inhibition of the growth of cancer cells, which is better than compound 6k (compound 25 in the reference cited above). In order to select and develop a leading compound among the quinoxaline compounds with substitutions on positions 5, 6, 7 or 8, the compounds comparable to compound 6k in in vitro cancer cell growth inhibition were chosen and their pharmacokinetic properties were evaluated in rats. In these studies, compound 6k showed the highest oral bioavailability of 83.4%, and compounds 6j and 6q followed, with 77.8% and 57.6%, respectively. From the results of in vitro growth inhibitory activities and the pharmacokinetic study, compound 6k is suggested for further development as an orally deliverable anticancer drug.

Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression.

A mixed culture of bacteria capable of growth on cyanide was isolated from an activated sludge of coal tar wastewater by an enrichment culture technique. The predominant cyanide-degrading microorganisms found in this bacterial mixture were identified as species of the genera Klebsiella, Serratia, Moraxella, and Pseudomonas. Stoichiometric amounts of ammonia were released during the cyanide containing culture by microbial oxidation of cyanide.

The AKT pathway is an important therapeutic target for cancer drug discovery as it functions as a main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers. In the present study, we found that an Akt1 antisense oligonucleotide (Akt1 AO) significantly downregulated the expression of AKT1 at both the mRNA and protein levels and inhibited cellular growth at nanomolar concentrations in various types of human cancer cells. Combined treatment of Akt1 AO with several cytotoxic drugs resulted in an additive growth inhibition of Caki-1 cells. The in vivo effectiveness of Akt1 AO was determined using two different xenograft nude mouse models. Akt1 AO (30 mg/kg, i.v. every 48 h) significantly inhibited the tumor growth of nude mouse subcutaneously implanted with U251 human glioblastoma cells after 27 days treatment. Akt1 AO (30 mg/kg, i.p continuously via osmotic pump) also significantly inhibited the tumor formation in nude mice implanted with luciferase-expressing MIA human pancreatic cancer cells (MIA-Luc) after 14 days of treatment. The luciferase signals from MIA-Luc cells were reduced or completely abolished after 2 weeks of treatment and the implanted tumors were barely detectable. Our findings suggest that Akt1 AO alone or in combination with other clinically approved anticancer agents should be further explored and progressed into clinical studies as a potential novel therapeutic agent.

Fluorocyclopentenylcytosine (RX-3117) is an orally bioavailable novel cytidine analog, currently being tested in a Phase I clinical trial. RX-3117 shows promising antitumor activity in various human tumor xenografts including patient derived xenografts resistant to gemcitabine. Initial characterization of RX-3117 indicates that this compound is incorporated into both RNA and DNA, and downregulates DNA methyltransferase I (DNMT1). RX-3117 is not deaminated by cytidine deaminase, an enzyme that limits the efficacy of most cytidine analogs due to extensive deamination. Our studies also demonstrate that RX-3117 is taken up by the equilibrative nucleoside transporter and is activated by uridine-cytidine kinase (UCK), since co-administration of uridine or cytidine protects cells against cytotoxicity of RX-3117, and inhibits RX-3117 phosphorylation in enzyme activity assays. Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation by transfecting two non-small cell lung cancer cell lines, A549 and SW1573 with siRNA directed against either UCK1 or UCK2 to determine whether downregulation would protect cells against RX-3117. UCK1-siRNA and UCK2-siRNA efficiently downregulated the expression of UCK1 and UCK2, respectively, in both cell lines, up to at least 72 hr after transfection. Exposure of UCK1-siRNA transfected cells to IC50 concentrations of RX-3117 did not affect the sensitivity of either A549 or SW1573 cells; however, transfection of UCK2-siRNA completely protected both A549 and SW1573 from the cytotoxic effects of RX-3117. In order to further investigate the role of UCK2, we tested the sensitivity of transfected cells to ethynylcytidine (ETC), a known substrate for UCK2. UCK2-siRNA transfection protected cells against ETC, but transfection with UCK1-siRNA did not. In order to explore whether we can use UCK2 expression in tumors as a predictive biomarker, we measured UCK enzyme activity, using radioactively labelled RX-3117 and the natural substrate uridine in 6 tumor cell lines to determine whether the enzyme activity correlates with UCK1 or UCK2 mRNA expression. UCK2 mRNA expression was significantly (p Citation Format: Godefridus J. Peters, Joris R. Julsing, Kees Smid, Daniel De Klerk, Dzjemma Sarkisjan, Mi Y. Yang, Young B. Lee, Deog J. Kim. Fluorocyclopentenylcytosine (RX-3117) is activated by uridine-cytidine kinase 2, a potential biomarker. [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 2622. doi:10.1158/1538-7445.AM2015-2622

The DEAD box RNA helicase DDX5/p68 is aberrantly expressed and post-translationally modified in several cancers. Several studies have indicated that p68 plays important roles in cell proliferation and tumor progression; in particular, p68 that is phosphorylated on Tyr593 has been shown to be associated with cell transformation, epithelial mesenchymal transition and cell migration. Therefore, p68 may be a promising target for novel anti-cancer therapeutics. We previously reported that 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl) aminocarbonyl] piperazine (RX-5902) inhibits the growth cancer cell lines by interacting and interfering with the function of phosphorylated p68 at low nanomolar concentrations, and inhibit tumor growth in several human xenograft models in nude mice. Although p68 is largely a nuclear protein, it has been found to shuttle between the nucleus and cytoplasm in cells, a function that appears to be critical for its role in epithelial-mesenchymal transition. Furthermore, our previous work demonstrated that the nuclear localization of p68 alters during the cell cycle suggesting that alterations in localization of p68 may also be important for cell cycle control and proliferation. We therefore investigated the effect of RX-5902 on the cellular localization of phosphorylated p68 in cancer cell lines. Our results suggest that treatment with RX5902 results in a significant alteration in the localization of phosphorylated p68, providing a possible mechanism(s) by which RX-5902 may affect phosphorylated p68 function. Further ongoing studies are aimed at examining the effect of RX-5902 on cellular motility in wound healing assays to determine whether RX-5902 could additionally affect the potential function of phosphorylated p68 in cell migration. Citation Format: Judit Remenyi, Mi Young Yang, Young Bok Lee, Deog Joong Kim, Frances V. Fuller-Pace. Targeting localization and function of the RNA helicase DDX5/p68 with 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl) aminocarbonyl] piperazine (RX-5902). [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 3577. doi:10.1158/1538-7445.AM2015-3577

RX-3117 (fluorocyclopentenylcytosine) is a novel small-molecule chemotherapeutic agent that belongs to the class of cytotoxic antimetabolite cytidine analogs. Cytidine analogs, including gemcitabine, have been widely used for the treatment of various types of cancer, both hematologic as well as solid. However, despite the success of gemcitabine, there is no oral formulation of gemcitabine and drug resistance is common. Thus, RX-3117 was synthesized as a oral formulation to overcome gemcitabine resistance with a better pharmacologic profile. In this study, the efficacy of RX-3117 was examined in 12 different human tumor (colon, non-small cell lung, small cell lung, pancreatic, renal, ovarian and cervical) xenograft models, grown subcutaneously in athymic nude mice. Not only has RX-3117 demonstrated potent efficacy in several cancer xenograft models but also oral treatment with RX-3117 results in dose-dependent tumor growth inhibition (TGI), even in tumors that are only moderately sensitive or resistant to gemcitabine. In the Colo-205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 14%, 25%, and 0% TGI, respectively, whereas oral treatment with RX-3117 induced 93%, 91%, 62%, and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. In order to extend the results established in cell line xenograft models, and to test efficacy in a potentially more clinically relevant system, RX-3117 was evaluated in a single primary low passage human pancreatic Tumorgraft™CTG-0298, which is resistant to gemcitabine and has a favorable RX-3117 activating enzyme profile. Treatment with RX-3117 resulted in dose dependent TGI and was superior to the standard of care agent gemcitabine. This study successfully demonstrated preliminary efficacy and therapeutic potential of RX-3117. Citation Format: Mi Young Yang, Young Bok Lee, Deog Joong Kim, Chang-Ho Ahn, Joel Kaye, Tania Fine, Rina Kashi, Osnat Ohne. A novel small molecule cytidine analog, RX-3117, shows potent efficacy in xenograft models, even in tumors that are resistant to treatment with gemcitabine. [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 819. doi:10.1158/1538-7445.AM2014-819

Over the last decade, it has been shown that nanopolymer therapeutics can be engineered to target cancer cells specifically and can deliver the cancer agent in a time-release fashion, thereby leaving normal healthy cells unaffected by toxic drugs such as docetaxel. Docetaxel has been used to treat several types of cancers, but it has provided pharmaceutical challenges due to its poor solubility and toxicities associated with the co-solvents. Given that nanotechnology can enhance the specificity, efficacy and safety of cancer agents and with the need of a new alternative formulation for docetaxel delivery, we developed a poly N-(2-hydroxypropyl) methacrylamide (HPMA)-Docetaxel (Doc) conjugate with Folate (FA) targeting ligand and evaluated the effect of the product in both in vitro human cancer cell lines and in vivo mice xenograft tumor models. Analysis of the product displayed the incorporation of 7.9% of docetaxel and 2.1% of folate with 38.6kDa MW and 1.92 of polydispersity, and an improvement in water solubility. The product inhibited the proliferation of a variety of human cancer cells in nM ranges in in vitro study. The maximal tolerated dose (MTD) of the product in mice was more than 150 mg/kg (as a docetaxel amount), which is much higher than that of free docetaxel (∼25 mg/kg). In mice bearing tumor xenografts, treatment with the polymer-conjugate strongly inhibited the growth of various human tumors, extended survival, and enhanced tumor regression significantly without effects on body weight compared to control animals. The results clearly demonstrate that our new polyHPMA-docetaxel-folate conjugate compound is a promising candidate for anti-tumor chemotherapeutics with reduced toxicity and prolonged survival. Citation Format: Young Bok Lee, Deog Joong Kim, Miyoung Yang, Chang-Ho Ahn, Anjan Nan. Synthesis of targeted docetaxel-polymer conjugate and its anti-tumor efficacy. [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 4485. doi:10.1158/1538-7445.AM2014-4485

In our previous study, we showed that a novel compound 1-(3,5-dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine induces apoptosis of cancer cells by downregulation of Bcl-2 protein levels and causing G2/M cell cycle arrest. We observed also a synergistic effect in growth inhibition when combined with known anticancer agents in cancer cells and potent anti-growth activity in drug-resistant cancer cells as well as anti-proliferation of cancer cells at IC50 values of low nanomolar concentrations. Oral administration of the compound led to inhibition of tumor growth and enhanced survival in several tumor xenograft models. Our recent studies indicated that the compound interacts with p68 RNA helicase (also known as DDX5), a member of the DEAD box family of RNA helicases. p68 has been demonstrated to play a vital role in cell proliferation and tumor/cancer progression. Our studies showed that the compound did not exert its anti-cancer effects by inhibition of RNA unwinding by p68. Instead, the compound completely abrogated the β-catenin stimulated ATPase activity of p68 with an IC50 of 61 nM. More extensive studies are ongoing to validate and further characterize our interesting discovery. Citation Format: Young Bok Lee, Deog Joong Kim, Gina Chun Kost, Julie Frank, Chang-Ho Ahn, Zhi-Ren Liu. Mechanistic study of a new 4-(3, 5-dimethoxyphenyl)-N-(7-fluoro-3-methoxyquinoxalin-2-yl)piperazine-1-carboxamide compound (RX-5902). [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 5507. doi:10.1158/1538-7445.AM2013-5507

To seek the possible molecular defect in a patient with deficient factor X plasma procoagulant activity, factor X gene exosn and splice junctions were subjected to heteroduplex analyses and sequencing. A mutation in exon 2 was confirmed as substitution of A by G at nucleotide position 206, coding for Gly instead of a Glu which is a normal precursor for gamma-carboxylated glutamic acid (Gla) at amino acid position 14. An abolished TaqI restriction site was used to indicate homozygosity of the defect, but occurrence of a gene deletion with attendant heterozygosity could not be excluded. The deletion of a Gla residue could affect the Ca(2+)-binding properties of factor X or confer a flexibility interfering with the interactive properties of the light chain. The defect could explain the decreased functional activity of circulating factor X and the mild bleeding tendency of the propositus.

Cytidine analogs play an important role in the treatment of various types of cancer, both solid tumors and leukemia. A novel cytidine analog fluorocyclopentenylcytosine (RX-3117) was characterized for its cytotoxic effects, its metabolism and its mechanism of action in a panel of 9 solid tumor and leukemic cell lines, as well as 6 variants resistant to gemcitabine, cytarabine and other pyrimidine analogs. Sensitivity in the parent cell lines after 72 hr exposure varied 75 fold with IC50 values from 0.4 to 30 µM RX-3117. The human equilibrative nucleoside transporter mediates transport of RX-3117, since its inhibition protected cells. Uridine and cytidine also protected cells against RX-3117, indicating that activation of RX-3117 is dependent on phosphorylation catalyzed by uridine-cytidine kinase (UCK), which was abundant in all tested cell lines, including the gemcitabine resistant variants. Deoxycytidine did not protect cells against RX-3117. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). After its uptake in cells, RX-3117 was rapidly metabolised to its nucleotides with the triphosphate being the most prominent form (90% of all nucleotides), while synthesis of the nucleotides was highest in the most sensitive cell lines (U937 and A2780 cells) and lowest in the least sensitive cells (CCRF CEM cells). No difference in nucleotide formation was observed between the SW1573 and its gemcitabine resistant variant SW1573/G. In the AG6000 cells, the dCK- variant of A2780 and resistant to gemcitabine and RX-3117, a normal monophosphate level was found, but no di-and triphosphates were formed, explaining its resistance. Similarly incorporation of RX-3117 into RNA and DNA was higher in the sensitive A2780 and low in the insensitive SW1573 cells, with no difference between the gemcitabine sensitive and resistant variants. The effect of RX-3117 on synthesis of RNA and DNA was quite different; in the sensitive U937 cells 10 µM RX-3117 inhibited RNA synthesis 90%, while in A2780 and CCRF-CEM cells 100 µM RX-3117 was required for 90% inhibition of RNA synthesis. The effect on DNA synthesis was quite different. 1 µM RX-3117 completely inhibited DNA synthesis in the sensitive U937 cells, 80-90% inhibition was achieved with 10 µM in both CCRF CEM variants and with 100 µM in SW1573/G and AG6000, but in A2780 and SW1573 cells 100 µM only resulted in a partial or no inhibition, respectively. In conclusion, RX-3117 showed a completely different sensitivity profile compared to other cytidine analogs. Its uptake is transporter dependent; it is not activated by dCK, but by UCK. RX-3117 is incorporated into RNA and DNA; RX-3117 hardly affected RNA synthesis at IC50 values, but inhibited DNA synthesis. Its metabolism to nucleotides is related with its sensitivity, possibly because they directly inhibit the target presumably DNA methyltransferase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1770. doi:1538-7445.AM2012-1770

Novel 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives were synthesized and evaluated as an anti-tumor agent. Despite their structural similarity to the quinoxalinyl-piperazine core scaffold, the IC50 values of the compounds against human cancer cells depends on the substitution at the quinoxaline ring but also from substituents at the phenyl ring. The best compound showing IC50 values ranging from 11 to 21 nμ was selected and characterized further both in vitro and in vivo. This compound was more potent against paclitaxel resistant HCT-15 colorectal cells compared to paclitaxel itself. Combined treatment of this compound with known anti-cancer drugs, such as paclitaxel, doxorubicin, cisplatin, gemcitabine or 5-fluorouracil, displayed synergistic growth inhibition of cancer cells. In mice bearing tumor xenografts, treatment with the compound completely inhibited the growth of various human tumors, enhanced tumor regression without effects on body weight compared to control animals. Mechanistic studies have shown that this quinoxalinyl-piperazine compound is a G2/M-specific cell cycle inhibitor and inhibits anti-apoptotic Bcl-2 protein with p21 induction. The results clearly demonstrate that our new quinoxalinyl-piperazine compound could become a novel class of anti-tumor chemotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1371. doi:10.1158/1538-7445.AM2011-1371

A series of isoquinolineamine small molecule derivatives were synthesized and screened as potential anticancer agents. Among them, we identified one isoquinolineamine analogue with potential anticancer activity. This compound inhibited the proliferation of a variety of cancer cells derived from many human solid tumors with IC50 values ranging from 14 nM to 71 nM. The compound also was more effective against gemcitabine and cisplatin-resistant cancer cell lines when compared to the original cytotoxic cancer drugs. In mice bearing tumor xenografts, treatment with the compound significantly inhibited the growth of tumors, enhanced tumor regression, and also prevented the growth of tumors in a paclitaxel-resistant xenograft model. This compound is well tolerated and had no effects on body weight compared to control animals. Mechanistic studies showed that cancer cells treated with this compound resulted in the elevation of cytochrome c in the cytosol, the decrease of Bcl-2 and the activation of Caspase 9. Further studies showed inhibition of the expression of several signaling molecules important for cell proliferation (p-Erk, p-p38 and p-Akt) and also decreased protein levels of p-GSK3α/β and β-catenin. Therefore, our result suggests that this compound could be a promising antitumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3562.