Hongnan Jin

IL-17E, a proinflammatory cytokine, has antitumor efficacy against several tumor types in vivo

Interleukin-17E (IL-17E) belongs to a novel family of cytokines that possess significant homology to IL-17. IL-17E has potent inflammatory effects in vitro and in vivo. Overexpression of IL-17E in mice results in a T helper-2 (Th2)-type immune response, which includes the expansion of eosinophils through the production of IL-5, and elevated gene expression of IL-4 and IL-13 in multiple tissues. In this study, we show that IL-17E has antitumor activity in vivo, a previously unrecognized function of IL-17E. Antitumor efficacy of IL-17E was examined in a variety of human tumor xenograft models, including melanoma, breast, lung, colon, and pancreatic cancers. Injection of recombinant IL-17E every other day resulted in significant antitumor activity in these tumor models. In addition, the combination of IL-17E with chemotherapy or immunotherapy agents showed an enhanced antitumor efficacy in human tumor xenograft models in mice as compared to either agent alone. Antitumor activity was demonstrated using different routes of administration, including intraperitoneal, intravenous, and subcutaneous injection. Anticancer activity was shown for both mouse and human forms of IL-17E, which have a high degree of sequence identity. Tumor-bearing mice treated with IL-17E showed a significant increase in serum levels of IL-5 and increased numbers of eosinophils in peripheral blood compared to the control group. Spleens isolated from IL-17E-treated mice showed a significant increase in eosinophils that correlated with antitumor activity of IL-17E in a dose–response manner. Finally, we demonstrate that B cells are necessary for IL-17E-mediated antitumor activity and that IL-17E was found to activate signaling pathways in B cells in vitro. Taken together, these data demonstrate that IL-17E has antitumor activity in vivo, and support further investigation of the potential clinical use of IL-17E as an anticancer agent.

A novel small molecule with potent anticancer activity inhibits cell growth by modulating intracellular labile zinc homeostasis

ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G1-S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development. [Mol Cancer Ther 2009;8(9):2586–96]

NK cell activation and tumor infiltration are involved in the antitumor mechanism of Virulizin

Previous studies have demonstrated antitumor efficacy of Virulizin in several human tumor xenograft models and a critical role for macrophages in the antitumor mechanism of Virulizin. Although there is growing support for an immune stimulatory mechanism of action for Virulizin, the details remain to be elucidated. The aim of this study was to determine whether infiltration of natural killer (NK) cells into xenografted tumors is altered by Virulizin treatment, and whether such alterations contribute to the antitumor activity of Virulizin. Immunohistochemical analysis demonstrated that xenografted tumors from Virulizin-treated mice had an increase in infiltration of F4/80+ (macrophages) and NK1.1+ (NK) cells. The increase in NK1.1+ cell infiltration occurred at an early stage of Virulizin treatment, which correlated with an early sign of apoptosis. In addition, Virulizin resulted in an increase in the number of NK cells in the spleens, and NK cells isolated from the spleen exhibited increased cytotoxicity to tumor cells in vitro. In NK cell–deficient SCID-beige mice, the antitumor activity of Virulizin was compromised, providing additional support to the hypothesis that NK cells are necessary for inhibition of tumor growth by Virulizin. Finally, depletion of macrophages resulted in the loss of Virulizin-induced increase in NK1.1+ cell infiltration into xenografted tumors, suggesting the involvement of macrophages in NK cell infiltration into tumors. Taken together, these results strongly support a mechanism in which Virulizin stimulates a sustained expansion and infiltration of NK cells and macrophages into tumors with subsequent activation of NK cells that is responsible for the observed antitumor activity.

Anti-proliferative and anti-tumor effects of antisense oligonucleotide GTI-2601 targeted against human thioredoxin.

Human thioredoxin has been implicated in cancer as a growth stimulator through regulation of DNA replication and growth factor activity, as a modulator of transcription factor activity, and as an inhibitor of apoptosis. In the present study, the steady-state level of thioredoxin protein was examined in a number of cancer cell lines. Interestingly, thioredoxin expression is elevated in a variety of human tumor cell lines compared with normal cell lines. The altered expression of thioredoxin in tumor cells suggests it may be a target in the development of novel therapeutic agents for the treatment and prevention of cancer. Further to this possibility, 26 phosphorothioate antisense oligodeoxynucleotides (PS-AS-ODNs) were evaluated for the ability to inhibit thioredoxin expression in cell culture. One PS-AS-ODN, GTI-2601, specifically reduced the levels of thioredoxin mRNA and protein, exhibited potent anti-proliferative effects on colony formation in vitro, and had anti-tumor effects in human tumor xenograft mouse models in vivo. Sequence-specific decreases in thioredoxin expression levels were accompanied by significant suppression of tumor growth in mice. Taken together, these data suggest that thioredoxin may be a useful target for developing PS-AS-ODNs as drug candidates against human cancer.

Abstract 4649: Utilization of KLF-4 as a pharmacodynamic biomarker for in vivo anticancer activity of a novel small molecule drug LOR-253.

LOR-253 is a potent and selective growth inhibitor of several cancer types, including non-small cell lung cancer (NSCLC), colon cancer and leukemia. LOR-253 stimulates Kruppel Like Factor-4 (KLF-4), a tumor suppressor factor which is characteristically deficient in a variety of cancers, and so represents a new approach to cancer therapy. The current Phase I dose-escalation study is being conducted at Memorial Sloan-Kettering Cancer Center in New York and MD Anderson Cancer Center in Houston. In the present preclinical study, the changes in KLF-4 expression levels were evaluated in vivo to examine the utility of KLF-4 as a pharmacodynamic biomarker, aiming at linking the antitumor effects of LOR-253 to the induction of KLF-4. In the human H226 NSCLC xenograft mouse model, LOR-253 demonstrated dose-dependent antitumor activity when administered at 1, 5, and 15 mg/kg and mediated significant tumor growth inhibition of about 55% at the highest dose given by intravenous bolus injection (p=0.05). Tumors were harvested 24h after the last dose following the treatments at 1, 5, and 15 mg/kg and KLF-4 mRNA and protein levels were analyzed by real-time RT-PCR and Western blot, respectively. A dose-dependent induction of KLF-4 mRNA and protein levels was observed. Pharmacodynamic studies to characterize the effect of LOR-253 on intratumoral KLF-4 were also conducted in the H226 NSCLC xenograft mouse model to determine the optimal time point for tumor biopsy sampling, following the treatment schedule in the ongoing Phase 1 clinical trial (2 days dosing, 12 day break, 2 days of dosing, 12 day break (1 cycle)). Tumor samples were taken 24h after the second, fourth and eighth dose of LOR-253 and analyzed for KLF-4 expression. It was determined that tumor sampling after the second and eighth doses was optimal at demonstrating increased intratumoral KLF-4 levels. Furthermore, antitumor efficacy of LOR-253 was also evaluated in nude rats bearing established human H226 NSCLC tumors when LOR-253 was infused intravenously following the current Phase I clinical dosing schedule. A significant efficacy was observed at the end of the first cycle of treatment, demonstrating 44% decrease in mean tumor volume when compared with the control treatment (p=0.025). Inhibition of tumor growth was paralleled by the induction of KLF-4 in isolated tumors when analyzed by real-time RT-PCR and Western blot. Taken together, the in vivo data obtained in both mouse and rat models provide strong preclinical support for utilizing KLF-4 as a potential pharmacodynamic biomarker for LOR-253 treatment in clinic. Citation Format: Howard Cukier, Robert Peralta, Hongnan Jin, Yu Cheng, Venkata Nedunuri, Salah Salehi, Yoon S. Lee, Aiping Young. Utilization of KLF-4 as a pharmacodynamic biomarker for in vivo anticancer activity of a novel small molecule drug LOR-253. [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 4649. doi:10.1158/1538-7445.AM2013-4649

Abstract 3710: Preclinical dose scheduling studies of LOR-253, a novel anticancer drug, in combination with chemotherapeutics in lung and colon cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL LOR-253 is an anticancer small molecule that is currently in a Phase I clinical study in patients with advanced or metastatic solid tumors. LOR-253 has a novel anticancer mechanism based on chelation of intracellular labile zinc, leading to inhibition of angiogenesis as well as G1/S cell cycle arrest due to induction of the tumor suppressor Kruppel-like factor 4 (KLF4). In preclinical studies, LOR-253 has shown potent antitumor activity against several cancers, including non-small cell lung cancer (NSCLC) and colon cancer, without significant toxicity. To support the clinical development of LOR-253 in combination with chemotherapeutics, we investigated the effect of dose scheduling of LOR-253 plus chemotherapy agents on anticancer activity in NSCLC (H226) and colon cancer (SW620) cell lines. Treatment of H226 cells in vitro with docetaxel, paclitaxel, or cisplatin for two days followed by increasing doses of LOR-253 resulted in significantly higher anti-proliferative activity than either agent alone. Assessment of drug interactions by determination of the combination index (D) (Berenbaum MC, Adv Cancer Res., 1981; 35:269-335) showed that the anticancer activities of the combination of LOR-253 with these chemotherapy agents were synergistic (D < 1). In studies with LOR-253 and docetaxel, synergistic anticancer activity against H226 was maintained with either concurrent or sequential treatments of each agent. Anticancer synergy was also seen in SW620 cells treated with LOR-253 in combination with oxaliplatin, CPT-11, or Fluorouracil. The effects of dose scheduling on activity of LOR-253 and docetaxel in vivo was also examined. Nude mice with H226 tumor xenografts were treated with repeat cycles of docetaxel once per week, followed two days later by treatment with LOR-253 for three consecutive days. Docetaxel was administered at an experimentally-determined ED50 dose level (defined as the effective dose at which 50% of mice showed tumor growth inhibition), while LOR-253 was given at its maximum efficacious dose in this model (10 mg/kg) or sub-optimal dose (5 mg/kg). Sequential administration of docetaxel followed by LOR-253 showed significant antitumor activity when docetaxel was given at either ED50 or sub-ED50 doses plus 10 mg/kg of LOR-253, compared to either agent alone given at the same dose, or with concurrent administration of LOR-253 and docetaxel at these dose levels. Preliminary results also show significant antitumor activity of LOR-253 plus docetaxel against H226 tumors in mice treated with repeat cycles of the reverse order of these treatments (LOR-253 for three days, followed by docetaxel 1X/week). In summary, dose scheduling studies with LOR-253 plus chemotherapy drugs demonstrate strong anticancer activities in NSCLC and colon cancer, providing support for the design of LOR-253 combination strategies for treatment of these cancers. 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 3710. doi:1538-7445.AM2012-3710

Abstract 667: Anticancer activity and tumor selectivity of LOR-253, a novel drug candidate, in lung carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL LOR-253 is a novel drug candidate entering a first in man dose escalation Phase I clinical trial in patients with advanced solid tumors. LOR-253 was well tolerated in single and repeat-dose GLP-toxicology studies conducted in rats and dogs at significantly higher doses than equivalent efficacious doses determined in xenograft models in mice, which indicates a potential wide therapeutic window. Previously we reported that LOR-253 reduced the levels of intracellular labile zinc leading to cell cycle arrest, through the down regulation of the metal-responsive transcription factor 1 (MTF-1) and the induction of the Kruppel like factor 4 (KLF4). KLF4 is a G1/S cell cycle checkpoint gene with tumor suppressor function in a variety of cancers. A recent study demonstrated dramatically decreased levels of KLF4 expression in most primary lung tumors (Hu et al. Clin Cancer Res 2009; 15 (18): 5688-95). Anti-tumor activity of KLF4 was demonstrated when KLF4 expression was enforced in cancer cell lines with low basal KLF4 expression levels. In addition, tumor growth in animals was highly compromised when KLF4 gene was stably transfected. The aim of this study was to investigate differential effects of LOR-253 on normal lung fibroblasts vs lung cancer cell lines with different basal levels of KLF4 expression. We found an inverse correlation between LOR-253 sensitivity and basal KLF4 expression. LOR-253 was highly active in three different NSCLC cell lines (H1299, H322 and H226) as shown by much lower IC50 values. KLF4 expression levels in these cell lines were significantly lower than that in normal cells. In contrast, tumor cell lines with KLF4 expression levels similar or higher than normal cells were less sensitive to LOR-253 treatment. Similar results were obtained in in vivo xenograft models, which showed higher in vivo efficacy of LOR-253 in treated mice harboring low basal KLF4-expressing tumors. A time-course gene and protein expression of KLF4 and cyclin D1 in normal lung fibroblast (HFL1) and in NSCLC H226 cells treated with LOR-253 demonstrated a differential effect between normal vs cancer cells. A maximum expression of KLF4 and suppression of cyclin D1 in H226 cells were observed between 16 and 24 hours of treatment while there was no significant effect on HFL1 cells. Based on these results the cell line H226 was selected for further LOR-253 target validation and efficacy biomarker expression studies. A dose-response effect of LOR-253 treatment on tumor growth and KLF4 and cyclin D1 expression was observed in the H226 xenograft model using RT-PCR, Western blot and immunohistochemical analysis. Studies are underway to determine the expression levels of other biomarkers such as MTF-1, HIF-1 a and b-catenin in response to LOR-253 treatment, as well as the effect of in vivo gene knock down and over-expression of MTF-1, KLF4 and other biomarkers on the antitumor activity of LOR-253. 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 667. doi:10.1158/1538-7445.AM2011-667