Eric B. Schwartz

The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity

BackgroundWe characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells.ResultsFLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO).ConclusionsThese data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

BackgroundCurcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.MethodsHuman and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.ResultsOur data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.ConclusionsThese data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound in vivo.

Structurally modified curcumin analogs inhibit STAT3 phosphorylation and promote apoptosis of human renal cell carcinoma and melanoma cell lines.

The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.

Small molecules, LLL12 and FLLL32, inhibit STAT3 and exhibit potent growth suppressive activity in osteosarcoma cells and tumor growth in mice

SummaryConstitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in osteosarcoma, and hence, may serve as a therapeutic target. In order to target STAT3, we tested two new STAT3 inhibitors, LLL12 and FLLL32. LLL12 and FLLL32 inhibit STAT3 phosphorylation and STAT3 downstream targets. LLL12 and FLLL32 also inhibit IL-6 induced STAT3 phosphorylation. The inhibition of STAT3 by LLL12 and FLLL32 resulted in the induction of apoptosis, reduction of plating efficiency, and migration in osteosarcoma cells. Furthermore, LLL12 and FLLL32 inhibited SJSA osteosarcoma cells and OS-33 tumor growth in murine xenografts. These results provide evidence that constitutive STAT3 signaling is required for osteosarcoma survival and migration in vitro and tumor growth in vivo. Blocking persistent STAT3 signaling by LLL12 and FLLL32 may be a novel therapeutic approach for osteosarcoma.

EGFR-STAT3 signaling promotes formation of malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) develop sporadically or in the context of neurofibromatosis type 1. Epidermal growth factor receptor (EGFR) overexpression has been implicated in MPNST formation, but its precise role and relevant signaling pathways remain unknown. We found that EGFR overexpression promotes mouse neurofibroma transformation to aggressive MPNST (GEM-PNST). Immunohistochemistry demonstrated phosphorylated STAT3 (Tyr705) in both human MPNST and mouse GEM-PNST. A specific JAK2/STAT3 inhibitor FLLL32 delayed MPNST formation in an MPNST xenograft nude mouse model. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR–STAT3 pathway is necessary for MPNST transformation and establishment of MPNST xenografts growth but not for tumor maintenance. Efficacy of the FLLL32 pharmacological inhibitor in delaying MPNST growth suggests that combination therapies targeting JAK/STAT3 might be useful therapeutics.

A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma.

Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. In this paper, a simple liquid chromatography-tandem mass spectrometric method was developed for the determination of these four curcuminoids in cell medium and mouse plasma. The method showed linearity from 1 to 1000 ng/mL with the lower limit of quantification of 1 ng/mL in cell medium, and 5 ng/mL in mouse plasma for all test curcuminoids. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85-115%. This method was subsequently used to evaluate their stability in these matrices and a pilot pharmacokinetics of curcumin, DMCHC and TMC in mice after an intraperitoneal (i.p.) cassette dosing of 10mg/kg each. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, THC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability is in the order curcumin<THC<TMC<DMCHC in both matrices. Following an i.p. cassette dose, both TMC and DMCHC showed the prolonged elimination half life (1.0, 1.0 h, respectively vs 0.4h for curcumin) and an increased drug exposure as described by the area under the curve (0.64, 0.98 μM h, respectively vs 0.4 μM h for curcumin).

Insertional mutagenesis identifies a STAT3/Arid1b/β-catenin pathway driving neurofibroma initiation

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

Inhibition of the JAK2/STAT3 signaling pathway exerts a therapeutic effect on osteosarcoma

Osteosarcoma (OS) is the most common type of malignant bone tumor. Despite aggressive multimodal treatments, including surgical resection, chemotherapy and adjunctive immunotherapies, patients with OS with high-grade malignancy have a poor five-year survival rate that has remained unchanged over the past two decades, highlighting the urgent requirement for novel therapeutic approaches. Signal transducers and activators of transcription 3 (STAT3) has been implicated as an oncogene and therapeutic target in a variety of neoplastic diseases. The aim of the present study was to determine whether inhibition of the janus kinase 2 (JAK2)/STAT3 pathway by FLLL32, a specific JAK2/STAT3 inhibitor, is able to provide a potential therapy for OS. FLLL32 inhibited OS cell growth in vitro and delayed OS growth in an OS xenograft nude mouse model. STAT3 knockdown by short hairpin RNA delayed OS formation in vivo. Thus, the JAK2/STAT3 pathway is important in OS formation. Efficacy of the FLLL32 pharmacological inhibitor in delaying OS growth suggests that targeting JAK2/STAT3 may be a potential therapeutic strategy for patients with OS.

Abstract 2937: An EGFR-STAT3 pathway promotes NF1 peripheral nerve tumorigenesis and transformation

Neurofibromatosis type 1 (NF1) is a very common inherited disease, affecting 1:3500 individuals worldwide. Nearly all (95%) of NF1 patients develop benign neurofibromas and malignant peripheral nerve sheath tumors(MPNSTs). Currently, their prevention is not possible, partially because the molecular mechanisms of tumorigenesis and the molecules that mark benign neurofibroma formation are poorly understood. This study is to test the relevance of EGFR expression to neurofibroma formation, and to identify possible additional pathways and genes that might contribute to neurofibroma formation. We bred the Nf1 flox/flox ;DhhCre mice, 100% of which form neurofibromas (Wu et al., 2008), to CNP-hEGFR mice and to Wa2 mice, an EGFR hypomorphic allele. To test the role of EGFR in tumorigenesis, we also used sleeping beauty (SB) insertional mutagenesis to obtain quadruple transgenic mice (Rosa26-lsl-SB11;T2/Onc; Nf1 flox/flox ;DhhCre). To define neurofibroma initiation and progression genes, we used Pyrosequencing to identify common insertion sites (CISs) that had more SB insertions that are most likely to harbor disease-related genes. We used ingenuity pathway analysis to predict pathways and genes that might contribute to neurofibroma formation. We used a “neurofibroma sphere” culture system, a method used for detecting self-renewing stem/progenitor cells, to determine inhibitory effects of a STAT3 inhibitor (FLLL32). We immunostained human and mouse sections with anti-pSTAT3 (tyr705) to determine STAT3 activation status. We found that mouse neurofibroma number and size increased in Nf1 flox/flox ;DhhCre mice with hEGFR expressed in nerve Schwann cells. Diminished EGFR signaling in Nf1 flox/flox ;DhhCre, Wa2/+ mice decreased neurofibroma number, not size. We used insertional mutagenesis to identify other modifiers of neurofibroma tumorigenesis. Analysis of CISs identified hubs involving GSK3B, TNF, and STAT3. STAT3 was the most significant changed pathway. Inhibition of STAT3 by shRNA or a specific STAT3 inhibitor FLLL32 blocked human neurofibroma-sphere formation. Immunohistochemistry identified STAT3(p705) in human and mouse neurofibromas and MPNSTs. FLLL32 inhibited cell proliferation and stimulated cell death as well as reduced neurofibroma growth in vivo in the Nf1 flox/flox ;DhhCre mouse neurofibromas. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR-STAT3 pathway regulates neurofibroma number and neurofibroma growth, and promotes transformation. STAT3 inhibitors may be useful in NF1 therapeutics. (*This work was supported by the National Institutes of Health (R01 NS28840 to N.R. and P50 NS057531 to N.R. and D.L.) and an Ohio State University Comprehensive Cancer Center pelotonia idea award to J.W.) 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 2937. doi:1538-7445.AM2012-2937

Cholangiocarcinoma cells secrete soluble factors that activate Jak/STAT signal transduction and promote MDSC expansion

Meeting abstracts Cholangiocarcinoma (CC) is a universally lethal disease that responds poorly to chemo- and immunotherapy. Patients with CC typically display elevated systemic interleukin-6 (IL-6) and systemic expansion of myeloid-derived suppressor cells (MDSCs). Interestingly, both of these