Luminescent Cell Viability Assay Kit Research Articles

Dexamethasone promotes osteoblast apoptosis through the Chk2/p53 signaling pathway.

Glucocorticoids (GCs) are widely used to treat inflammatory or autoimmune diseases. However, several studies have reported that the use of GCs can lead to numerous complications, the most serious of which are osteoporosis and osteonecrosis of the femoral head (ONFH). Osteoblast apoptosis has been identified as an important event in the development of GC-induced osteoporosis and ONFH. However, the mechanisms underlying the regulation of these processes have not yet been explored. To observe the effect of dexamethasone (Dex) on the apoptosis of osteoblasts and explore its mechanism, as well as provide a new therapeutic idea for GC‑induced osteoporosis and ONFH. Cell proliferation and apoptosis of MC3T3-E1 cells after Dex treatment were determined using the CellTiter-Glo® Luminescent Cell Viability Assay kit and Annexin V-FITC/PI Double Staining Apoptosis Detection Kit, respectively. The expression of caspase-3/cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP)/cleaved PARP in MC3T3-E1 cells after Dex treatment was determined with western blotting. The expression of p53 and checkpoint kinase 2 (Chk2) in MC3T3-E1 cells after Dex treatment was analyzed using western blotting and polymerase chain reaction (PCR). The effects of p53 knockdown and Chk2 knockdown on Dex-induced apoptosis of MC3T3-E1 cells were also characterized. Dexamethasone remarkably inhibited cell growth and induced the apoptosis of MC3T3-E1 cells. We also observed that Dex induced osteoblast apoptosis by promoting p53 expression. The regulatory effect of Dex on p53 expression is mediated by the upregulation of Chk2, which interacted with p53 and inhibited p53 degradation. The knockdown of p53 alleviated Dex-induced MC3T3-E1 cell apoptosis by decreasing the expression of cleaved caspase-3 and cleaved PARP. We demonstrated that Dex increased Chk2 protein expression, which stabilized the protein expression of p53, and in turn promoted osteoblast apoptosis.

EXTH-57. EFFICACY OF SOLUBLE PANOBINOSTAT (MTX110) IN PRECLINICAL MODELS OF ADULT GLIOBLASTOMA

Abstract INTRODUCTION One of the biggest challenges in treating glioblastoma is achieving effective local drug concentrations, and trials using systemic administration of therapeutics have failed in GBM despite compelling pre-clinical evidence. MTX110 (Midatech Pharma PLC) is a water-soluble form of panobinostat currently in clinical development for DIPG and medulloblastoma using direct tumor delivery. We have previously reported a significant positive benefit of MTX110 in a subcutaneous GBM mouse model in an IDH1 mutated cell line. Here we present follow on data on the potential for MTX110 synergy with radiation in pre-clinical in vivo models. We also present data on efficacy of MTX110 in a panel of GBM cell lines. METHODS In vitro: Cell lines were incubated with MTX110 for 72h over a range of concentrations (1nM-10µM). Each concentration was tested in triplicate with the appropriate blank controls included in each plate. After 72 hours, cell viability was measured via luminescence signal (Promega CellTiter-Glo Luminescent Cell Viability Assay kit (Promega-G7573) read via 2104 EnVision Multilabel Reader, PerkinElmer. In vivo: Tumor-bearing mice were stratified to either the vehicle control or treatment group based on median tumor volume and were treated with MTX110 at a dose of 15mg/kg IP, 5 days consecutively for 2 consecutive weeks, following radiation treatment at a dose of 4Gy delivered as a single fraction on day 1. RESULTS In vitro, MTX110 demonstrated cytotoxic activity in 4 GBM cell lines (U87MG, U251MG, U118MG and T98G) with an average IC50 value in the region of 40nM (17-43nM). In vivo MTX110 in combination with radiation treatment showed an enhanced delay in tumor growth of approximately 50% compared to MTX110 or radiation alone. The results are supportive of the planned exploratory trial in GBM with MTX110.

Identify effects of phenytoin and losartan on TGF‐beta using human gingival fibroblast‐1 (HGF‐1) cells and establish HGF‐1 cell culture as a model to study drug‐induced gingival hyperplasia

PurposeThe main objectives of this study were 1) to evaluate the effects of phenytoin (antiepileptic drug) and losartan (TGF‐beta inhibitor) on cytotoxicity, ATP, and TGF‐beta in human gingival fibroblasts (HGF‐1) cells, 2) to identify whether losartan inhibits gingival overgrowth caused by phenytoin and 3) to establish HGF‐1 cell culture as a model to study druginduced gingival hyperplasia.MethodsHGF‐1 cells obtained from ATCC were grown to 90% confluency in DMEM media supplemented with PenStrep, and bovine serum (FBS 10%). Cells were incubated in 5% CO2, 37°C and 95% relative humidity. For cytotoxicity, LDH leakage in the medium was measured using the CytoTox 96 Non‐radioactive Cytotoxicity Assay Kit, and for ATP, total % ATP was measured using CellTiter‐Glo Luminescent Cell Viability Assay Kit. For ATP and LDH measurements, cells were treated with various concentrations of phenytoin (0, 10, 25, 50, 100 and 200 μM) and losartan (0, 25, 50, 100, 250 and 500 μM) dissolved in DMSO, and incubated for 24h (30,000 cells/well in 96 well plates), 5% CO2, 37°C and 95% relative humidity. Final DMSO concentration was 0.1%. To identify effects on TGF‐beta, cells were treated with DMSO (0.1% final concentration), Phenytoin (100 μM), Losartan (100 μM), and Phenytoin (100 μM) + Losartan (100 μM). After 24h treatment of cells, TGF beta was measured as per supplier’s protocol using ELISA assay kit (Thermo Fisher, Grand Island, NY). All experiments were carried out as triplicate measurements and mean of triplicates were considered as one experiment (n). Minimum of three repetitions (n = 3) were carried out unless otherwise noted in results.ResultsPreliminary results show that none of the drug concentrations had a cytotoxic effect on HGF‐1 cells. No increase in ATP levels were observed for losartan. However, phenytoin showed greater increase in percent ATP levels when compared to vehicle control. Percent ATP levels for losartan and phenytoin were in the range of 101 ± 4 to 113.5 ± 8.2% and 135 ± 11.1 to 138.1 ± 16.5% respectively. Data from one TGF‐beta experiment showed no increase in TGF‐beta levels with phenytoin treatment, however losartan, and losartan plus phenytoin treatment showed 1.3 and 2‐fold increase in TGF beta levels over untreated cells respectively.ConclusionOur study suggests that phenytoin causes cell proliferation as noted from increased ATP levels. Preliminary results form TGF‐beta experiments were inconclusive and further experiments are needed to ascertain effects of losartan and phenytoin on TGF‐beta in HGF‐1 cells and identify whether HGF‐1 cells can be used as a model to study drug‐induced gingival hyperplasia.Support or Funding InformationThis project is supported by College of Pharmacy, Roseman University of Health Sciences, South Jordan, Utah 84095

Abstract A018: Synergy between IAP inhibitors and a cytotoxic antibody-based chimeric protein in pancreatic cancer cells

Abstract Background: immunotoxins are cytotoxic antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. Immunotoxins derived from Pseudomonas exotoxin inhibit protein synthesis via the ADP- ribosylation of the eukaryotic elongation factor (eEF2). Clear clinical benefit has been achieved in patients treated with an immunotoxin targeting CD22 expressed on B-cell malignancies and the immunotoxin has recently been approved by FDA. However, success has not been universal and, when treating solid tumors, immunotoxins have performed less well in producing complete responses, due to poor apoptotic responses. Screening for enhancing compounds revealed IAP inhibitors as potentially useful for improving immunotoxin killing. The immunotoxin, LMB-100, targets surface mesothelin on pancreatic tumor cells but when tested on two pancreatic adenocarcinoma cell lines, AsPC1 and BxPc3, there was no detectable reduction in cell viability. Upon further investigation, we found that birinapant, an IAP inhibitor, strongly synergized with this immunotoxin against AsPC1 but not against BxPC3 cells. We sought to understand the different responses in each cell line. Materials and Methods: viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison WI). TNFalpha in the cell medium was detected using the hTNFa QuantiGlo ELISA (R&D System). The level of the major cell-associated apoptotic proteins was assessed using the Human apoptosis Array kit (R&D System). The spots on the arrays were quantified using ImageJ. Results: both AsPC1 and BxPC3 cells were resistant to the immunotoxin LMB-100. In the case of AsPC1, the immunotoxin caused only a modest cytostatic response at high immunotoxin concentrations at 72h whereas, in combination of birinapant, there was a strong cytotoxic effect, starting at 48h. BxPC3 cells remained resistant to the immunotoxin even in the presence of birinapant: the combination with the highest concentrations of both compounds reduced cell viability only to 50% after 72 hours. From the evaluation of the level of the major apoptotic proteins, we found that the most significative difference between the two cell lines was the phosphorylated status of p53, in particular the S392 hyper-phosphorylation in BxPC3 which has been associated with a poor prognosis, advanced tumor stage and grade in p53 positive cancers. AsPC1 cells also had a higher level of Bax, making them more predisposed to apoptosis. No difference in TNFalpha secreted in the medium or TNFR1 protein level was detected between the two cell lines. Conclusion: knowing the level of key proteins involved in the cell death pathway can explain the efficacy of drug combinations and could be used to predict responses. Citation Format: Antonella Antignani, David J FitzGerald. Synergy between IAP inhibitors and a cytotoxic antibody-based chimeric protein in pancreatic cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A018. doi:10.1158/1535-7163.TARG-19-A018

Taxol-Resistant Gene 1 (Txr1) Mediates Oxaliplatin Resistance by Inducing Autophagy in Human Nasopharyngeal Carcinoma Cells.

BackgroundOxaliplatin (L-OHP) is an important chemotherapy regimen for nasopharyngeal carcinoma (NPC), but can fail due to drug resistance. In this study, the role of Txr1 (taxol-resistant gene 1) in oxaliplatin resistance was investigated.Material/MethodsCell viability assay was carried out using the CellTiter-Glo Luminescent Cell Viability Assay Kit. CNE1 and CNE2 cells were cultured continuously with gradually increasing concentrations of L-OHP for 6 months to establish drug-resistant cell lines. Autophagy was detected by electron microscopy. Txr1 expression in NPC cells was detected via Western blotting and real-time quantitative PCR (qRT-PCR).ResultsIn L-OHP-resistant CNE1/L-OHP and CNE2/L-OHP cells, mRNA and protein expression of Txr1 increased compared to the parental cells, and downregulation of Txr1 re-sensitized drug-resistant cells to L-OHP. Moreover, we found that Txr1-mediated L-OHP resistance was associated with increased autophagy. Txr1-overexpression cells developed L-OHP resistance and a high level of autophagy. Inhibiting autophagy using 2 different methods – inhibition of autophagy-related gene expression and autophagy inhibitor – attenuated L-OHP resistance of NPC cells.ConclusionsWe conclude that the detection of Txr1 might become a good indicator to evaluate the treatment and prognosis of nasopharyngeal carcinoma. Our data suggest that further investigation of Txr1 in the setting of L-OHP resistance is warranted.

Identification of Tipifarnib Sensitivity Biomarkers in T-Cell Tumor Cell Lines

BackgroundT-cell leukemia and lymphoma (TCL) is a very aggressive and heterogeneous group of hematological malignancies with poor prognosis and inadequate response to current therapies. The standard first-line treatments have resulted in unsatisfactory patient outcomes and unfortunately, targeted treatments are still at a preliminary phase. The RAS/MAPK pathway is crucial for TCR signaling of T-cells and it is deregulated in TCL. We aimed to determine the therapeutic value of farnesyl transferase inhibitor (FTIs) tipifarnib in TCL cell lines. Effects on cell viability, apoptosis, cell cycle and gene expression were evaluated. Tipifarnib blocks the localization of some farnesylated proteins, including some RAS family members, to cellular membranes, thereby inhibiting their activation. Tipifarnib is a potent and specific FTI with prominent anti-proliferative effects.MethodsWe tested tipifarnib in 18 T-cell leukemia and 4 T-cell lymphoma cell lines for in vitro sensitivity and for biomarker discovery, both genomic and immunohistochemical. We selected cell lines with available genomic data from COSMIC, CCLE or generated by our group. The MAPK, NFAT, NFKB and JAK/STAT pathways were tested by immunohistochemistry analysis under basal conditions. The range of drug concentrations to perform IC50 analysis was established between 0-10,000 nM (ten points), using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) following manufacturer´s instructions at 0h, 48h and 96h. All experiments were done in sextuplet and all numerical data were expressed as the average of the values ± the standard error of the mean. IC50 analyses were performed with GraphPad Prism v5. Clinically-relevant drug sensitivity was defined as IC50 <100nM at 96h (defined as sensitive). RNAseq was performed in 4 sensitive cell lines with tipifarnib and DMSO (vehicle) in three biological replicates at 72h. Differential gene expression (DE) analysis was carried out under these two conditions. Pathway analysis was done using Gene Set Enrichment Analysis (GSEA). Induction of apoptosis and cell viability were tested by flow cytometry in sensitive cell lines.Results59.1% (n=13) of cell lines were sensitive to tipifarnib at concentrations which are readily achievable in the clinic. RAS, RAS-guanine nucleotide exchange factors (GEFs) and RAS-GTPase activating proteins (GAPs) genes were mutated in 45.5%, 50% and 27.3%, of cell lines respectively, but their mutational status was not associated with drug sensitivity. We found TP53 and DNMT3 mutated in 80% and 33.3% in sensitive cell lines respectively, and in 63.6% and 0% in resistant cell lines. The mutational state of NOTCH1 was associated with tipifarnib sensitivity; 66.7% in sensitive cell lines versus 9.1% in resistant cell lines (p=0.005). ERK activation (MAPK pathway) was significantly associated with drug sensitivity (p=0.046). Conversely, RelB (NFκB pathway) was associated with drug resistance (p=0.007). GSEA analysis showed that tipifarnib downregulated cell cycle, protein localization to membranes, metabolism, and ribosome and mitochondrial activity. The effect of tipifarnib on cell cycle progression and apoptosis was validated by flow cytometry assays. Tipifarnib decreased cellular viability, increased apoptosis and blocked cell cycle progression in G1 phase. GSEA of molecular pathways regulated by tipifarnib identified downregulation of MYC targets and mTOR and MAPK pathways. MYC is a target of NOTCH1, mutated in sensitive cases and associated with tipifarnib sensitivity.ConclusionsIn vitro response to tipifarnib in T-cell lymphoma and leukemia cell lines can be predicted using several biomarkers. The mutational NOTCH1 status, together with p-ERK (MAPK activation) were associated with increased sensitivity while RelB expression was associated with resistance. DisclosuresGualberto:Kura Oncology: Employment, Equity Ownership. Piris:Takeda: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Kura: Honoraria.

Overexpression of lncRNA IGFBP4\u20131 reprograms energy metabolism to promote lung cancer progression

BackgroundReprogrammed energy metabolism as an emerging hallmark of cancer has recently drawn special attention since it facilitate cell growth and proliferation. Recently, long noncoding RNAs (lncRNAs) have been served as key regulators implicated in tumor development and progression by promoting proliferation, invasion and metastasis. However, the associations of lncRNAs with cellular energy metabolism in lung cancer (LC) need to be clarified.MethodsHere, we conducted bioinformatics analysis and found insulin-like growth factor binding protein 4–1 (IGFBP4–1) as a new candidate lncRNA located in the upstream region of IGFBP4 gene. The expression levels of lnc-IGFBP4–1, mRNA levels of IGFBP4 in 159 paired lung cancer samples and adjacent, histological normal tissues by qRT-PCR. Over-expression and RNA interference (RNAi) approaches were adopted to investigate the biological functions of lnc-IGFBP4–1. The intracellular ATP level was measured using the Cell Titer-Glo Luminescent Cell Viability Assay kit, and changes in metabolic enzymes were examined in cancer cells and normal pulmonary epithelial cells with qRT-PCR.ResultsOur results showed that lnc-IGFBP4–1 was significantly up-regulated in LC tissues compared with corresponding non-tumor tissues (P < 0.01), and its expression level was significantly correlated with TNM stage (P < 0.01) and lymph node metastasis (P < 0.05). Further investigation showed that overexpression of lnc-IGFBP4–1 significantly promoted LC cell proliferation in vitro and in vivo, while downregulation of endogenous lnc-IGFBP4–1 could inhibited cell proliferation and induce apoptosis. Moreover, we found lnc-IGFBP4–1 could influences ATP production levels and expression of enzymes including HK2, PDK1 and LDHA, in addition, decline in both ATP production and these enzymes in response to 2-DG and 2-DG-combined Rho123, respectively, was observed in lnc-IGFBP4–1-overespressing LC cells, indicative of an enhanced aerobic glycolysis rate. Finally, lnc-IGFBP4–1 was observed to negatively correlate with gene IGFBP4, and lower expression level of IGFPB4 was found after lnc-IGFBP4–1-overexpression was transfected into PC9 cells, higher expression level of IGFPB4 was also found after lnc-IGFBP4–1-downregulation was transfected into GLC-82 cells, which indicates that IGFBP4 may exert its targeting function regulated by lnc-IGFBP4–1.ConclusionsTaken together, these findings provide the first evidence that lnc-IGFBP4–1 is significantly up-regulated in LC tissues and plays a positive role in cell proliferation and metastasis through possible mechanism of reprogramming tumor cell energy metabolism, which suggests that lnc-IGFBP4–1 may be a promising biomarker in LC development and progression and as a potential therapeutic target for LC intervention.

Identification of an easy to use 3D culture model to investigate invasion and anticancer drug response in chondrosarcomas

BackgroundCytotoxic efficacy of anticancer drugs has been widely studied with monolayer-cultured cancer cells. However, the efficacy of drugs under two-dimensional (2D) culture condition usually differs from that of three-dimensional (3D) one. In the present study, an in vitro tumor tissue model was constructed using alginate hydrogel, and in vitro cytotoxic efficacy of two anticancer drugs (cisplatin and DZNep) was investigated in chondrosarcomas, and compared to in vivo response.MethodsThree cell lines derived from human chondrosarcomas, CH2879, JJ012 and SW1353, were embedded in alginate hydrogel. Proliferation and survival were assayed by ATP measurement using Cell Titer-Glo luminescent cell viability assay kit, and by counting viable cells in beads. Collagen and COMP expression was determined by RT-PCR. Invasion/migration was estimated by counting cells leaving alginate beads and adhering to culture dish. Then, chondrosarcoma response to cisplatin and DZNep was compared between cells cultured in monolayer or embedded in alginate, and using chondrosarcoma xenografts in nude mice.ResultsChondrosarcomas survived at least for 8 weeks, after embedment in alginate. However, only CH2879 cells could proliferate. Also, this cell line is more invasive than SW1353 and JJ012, which was coherent with the grade of their respective primary tumors. Furthermore, the expression of type II collagen was higher in chondrosarcomas cultured in 3D than in 2D. Interestingly, this 3D culture system allows to validate the absence of response of chondrosarcomas to cisplatin, and to predict the efficiency of DZNep to reduce chondrosarcoma growth in vivo.ConclusionsThis study validates alginate beads as a relevant 3D model to study cancer biology and tumor responses to biological treatments.

Serum and tumor tissue microRNA-23a expression level in glioblastoma patients and the regulation mechanism of ATP5A1 expression by microRNA-23a

Objective To test the serum and tumor tissue expression level of microRNA (miRNA, miR)-23a in glioblastoma (GBM) patients and the functional role of miR-23a in GBM. Methods Expression level of serum and tumor tissue miR-23a in GBM patients was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). U87MG, U251, T98G and A172 cells were transfected with miR-23a mimic and the cell proliferation assay was done by CellTiter-Glo® Luminescent Cell Viability Assay kit, at the same time, the tumor growth of U87MG and U251 cells transfected with miR-23a mimic was measured. The expression level of ATP5A1 in GBM tumor tissue was evaluated by RT-qPCR and immunostaining. U87MG, U251, T98G and A172 cells were transfected with miR-23a mimic and the ATP5A1 protein expression was measured by Western blotting. Results The miR-23a expression level in serum (0.40±0.18) and tumor tissue (1.00±0.12) of GBM patients was downregulated compared with health control and tumor adjacent tissue (2.60±0.45 and 1.50±0.56, P=0.005); the miR-23a mimic could suppress the cell growth of U87MG, U251, T98G and A172 cells in vitro (P=0.011), tumor growth of U87MG and U251 cells in vivo, tumor volume of pLKO.1 infected U87MG and U251 was (597.1±95.4) cm3 and (429.5±78.7) cm3, tumor volume of miR-23a mimic infected U87MG and U251 was (153.2±38.6) cm3 and (168.7±33.1) cm3(P=0.008); RT-qPCR and Western blotting results revealed that ATP5A1 (5.4±1.1) was upregulated in GBM tumor tissue, and miR-23a mimic could suppress the protein expression level of ATP5A1 in U87MG, U251, T98G and A172 cells. Conclusion miR-23a could suppress the proliferation and tumor growth of GMB cell lines. Besides it could regulate the protein expression of ATP5A1. Key words: Gioblastoma; MicroRNA-23a; ATP5A1

Elucidate the Role of TGF‐beta in Prevention and Treatment of Gingival Hyperplasia Using HGF‐1 Cells

PurposeThe purpose of this study was to evaluate the effects of nifedipine (calcium channel blocker), phenytoin (antiepileptic drug), and losartan (TGF‐beta inhibitor) on cytotoxicity and ATP levels in human gingival fibroblasts (HGF‐1) and evaluate whether losartan attenuates gingival overgrowth caused by nifedipine and phenytoin.MethodsHGF‐1 cells obtained from ATCC were grown to confluency in D‐MEM media supplemented with PenStrep, and bovine serum (FBS 10%) in a CO2 incubator in T‐flasks. For cytotoxicity (LDH leakage in the medium was measured using the CytoTox 96 Non‐radioactive Cytotoxicity Assay Kit) and ATP (Total % ATP was measured using CellTiter‐Glo Luminescent Cell Viability Assay Kit). For ATP and LDH measurements, cells were treated with various concentrations of nifedipine (5, 10, 25, 50 and 100 μM), phenytoin (10, 25, 50, 100 and 200 μM) and losartan (25, 50, 100, 250 and 500 μM) dissolved in DMSO and incubated for 24 h, 72 h and 7 day periods (1×104 cells/well in 96 well plates, 5% CO2, 37 °C and 95% relative humidity).ResultsResults show that none of the drug concentrations had a significant cytotoxic effect on HGF‐1 cells. No significant increase in ATP levels were observed for losartan. At 24h, nifedipine and phenytoin showed significant increase in percent ATP levels when compared to vehicle control. Percent ATP levels for losartan, nifedipine and phenytoin were in range of 101 ± 4 to 113.5 ± 8.2%; 143.1 ± 16.9 to 148.5 ± 20.4% and 135 ± 11.1 to 138.1 ± 16.5% respectively after 24h treatment.ConclusionIn conclusion, our study suggests that nifeidipine and phenytoin causes cell proliferation as evidenced from increased ATP levels. Concentrations identified from these studies will be used to assess effects of losartan on nifedipine and phenytoin induced gingival overgrowth using various markers involved in gingival overgrowth.Support or Funding InformationThis project is supported by College of Pharmacy, Roseman University of Health Sciences, South Jordan, Utah

Targeting Protein Arginine Methyltransferase 1 Blocks RBM15-MKL1 Fusion-Initiated Human Stem Cell Transformation

Objective: The chromosome translocation t (1;22), which generates the RBM15-MKL1 fusion gene, is found in approximately 10% of pediatric acute megakaryoblastic leukemia cases. Given that PRMT1 downregulation is critical for megakaryocyte differentiation, we propose the use of the PRMT1 inhibitor furamidine to stimulate RBM15- MKL1-transformed human cord blood cells to undergo megakaryocyte differentiation. Materials and methods: Human CD34+ cells were purified from umbilical cord blood with anti-CD34 magnetic beads. Lentivirus-infected CD34+ cells were sorted using flow cytometry. The methylcellulose colony replating assay was performed to evaluate the transformation efficiency. Cell viability was calculated using a CellTiter-Glo® luminescent cell viability assay kit. Results: The simultaneous transduction of RBM15-MKL1 and MPLW515L, a mutated MPL gene found in AMKL patients, into human CD34+ cells resulted in long-term growth in the presence of a cytokine mix that maintains a population of hematopoietic stem progenitor cells. Elevated expression of PRMT1 was detected in cells transduced with RBM15-MKL1 together with MPLW515L. The PRMT1 inhibitor furamidine (aka DB75) reversed the inhibition of RBM15-MKL1-mediated megakaryocytic differentiation and impeded the replating capability of the transformed cells. Conclusion: PRMT1 facilitates the transformation induced by RBM15-MKL1, and inhibiting PRMT1 activity promotes MK differentiation. Given that furamidine has been used for treating trypanosomiasis in clinical trials and proven to be safe, using furamidine to treat AMKL may be a new curative option for RBM15-MKL1-associated leukemia.

Abstract 198: The phosphatase PRL-3 is expressed in primary B-ALL cells and is important for adhesion and migration

Abstract Introduction: Phosphatase of regenerating liver -3 (PRL-3) is a dual specificity phosphatase, and is associated with aggressiveness and metastatic disease in a number of solid tumors. Less is known about its role in hematological cancer, but we have previously shown that PRL-3 is expressed in primary multiple myeloma (MM) cells and MM cell lines. More recently PRL-3 is shown to be expressed in acute myeloid leukemia (AML), and associated with poor prognosis. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite progress in treatment and survival, treatment related toxicity is a major concern. Therefore, it is a need for new clinical markers and therapy targets to optimize the treatment. We hypothesized that PRL-3 is expressed, and have oncogenic functions in B-ALL, and can be a possible clinical marker and a novel drug target. Methods: Primary ALL cells from peripheral blood were obtained from The Regional Biobank of Central Norway, and PRL-3 mRNA expression was measured with qRT-PCR. The PreB-ALL cell lines Reh and MHH-CALL-4 were used for further experiments. IL-7 (100 ng/mL) were used for stimulation. ShRNA against PRL-3 was used to knock down PRL-3 in Reh (shPRL-3) and for empty vector control (shMOCK) by use of lentiviral transduction. To study adhesion we used fibronectin (20 μg/mL) and stimulated with IL-7, IL-8 and IGF-1 (all 100 ng/mL) for 45 min in 37°C. Migration was studied with transwell permeable plates (pore size 3 μm) using SDF1α (75 ng/mL) as a chemoattractant. Further we examined the effect of the small molecular inhibitor PRL-3 Inhibitor I on ALL cell lines. Apoptosis and cell viability was determined by Annexin-FITC/-PI with flow cytometry, and by CellTiter-Glo® Luminescent Cell Viability assay kit, after 24-72 hours incubation with PRL-3 Inhibitor I. Results: 89% (16 of 18) of primary B-ALL samples expressed PRL-3, and 33% (6 of 18) at high levels. Stimulation with IL-7 induced expression of PRL-3 in a dose-dependent manner in Reh and MHH-CALL-4. The knock down of PRL-3 by shRNA was 80% efficient. Reh shMOCK cells were significantly more adherent to fibronectin after stimulation with IL-7, IL-8 and IGF-1 compared to Reh shPRL3. The cells’ ability to migrate towards a SDF1α gradient were significantly reduced in Reh shPRL-3 cells compared with Reh shMOCK. PRL-3 inhibitor I also reduced the viability and induced apoptosis in Reh cell line in a dose-dependent manner, but not in MHH-CALL-4 cell line, at a concentration of 40-80 μM. Conclusion: PRL-3 was expressed in 89% of primary B-ALL samples. Knock down of PRL-3 clearly reduced cell adhesion and migration. PRL-3 inhibitor I induced apoptosis and reduced cell viability in Reh, but not in MHH-CALL-4. This study indicates that PRL-3 could be important in the pathogenesis of B-ALL, and might be a suitable target for treatment in the future. Citation Format: Magnus Aassved A. Hjort, Pegah Abdollahi, Esten Vandsemb, Tobias Schmidt Slørdahl, Bendik Lund, Magne Børset, Torstein Baade Rø. The phosphatase PRL-3 is expressed in primary B-ALL cells and is important for adhesion and migration. [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 198.

Antitumor Activity of HM781-36B, alone or in Combination with Chemotherapeutic Agents, in Colorectal Cancer Cells.

PurposeHM781-36B is a novel and irreversible pan-human epidermal growth factor receptor (HER) inhibitor with TEC cytoplasmic kinase inhibition. The aim of this study is to evaluate the antitumor activity and mechanism of action for HM781-36B in CRC cell lines.Materials and MethodsThe CRC cell lines were exposed to HM781-36B and/or oxaliplatin (L-OHP), 5-fluorouracil (5-FU), SN-38. The cell viability was examined by Cell Titer-Glo luminescent cell viability assay kit. Change in the cell cycle and protein expression was determined by flow cytometry and immunoblot analysis, respectively. Synergism between 2 drugs was evaluated by the combination index.ResultsThe addition of HM781-36B induced potent growth inhibition in both DiFi cells with EGFR overexpression and SNU-175 cells (IC50 = 0.003 and 0.005 μM, respectively). Furthermore, HM781-36B induced G1 arrest of the cell cycle and apoptosis, and reduced the levels of HER family and downstream signaling molecules, pERK and pAKT, as well as nonreceptor/cytoplasmic tyrosine kinase, BMX. The combination of HM781-36B with 5-FU, L-OHP, or SN-38 showed an additive or synergistic effect in most CRC cells.ConclusionThese findings suggest the potential roles of HM781-36B as the treatment for EGFR-overexpressing colon cancer, singly or in combination with chemotherapeutic agents. The role of BMX expression as a marker of response to HM781-36B should be further explored.

Histone deacetylase 6 activity is critical for the metastasis of Burkitt’s lymphoma cells

BackgroundBurkitt’s lymphoma is an aggressive malignancy with high risk of metastasis to extranodal sites, such as bone marrow and central nervous system. The prognosis of metastatic Burkitt’s lymphoma is poor. Here we sought to identify a role of histone deacetylase 6 (HDAC6) in the metastasis of Burkitt’s lymphoma cells.MethodsBurkitt’s lymphoma cells were pharmacologically treated with niltubacin, tubacin or sodium butyrate (NaB) or transfected with siRNAs to knock down the expression of HDAC6. Cell migration and invasion ability were measured by transwell assay, and cell cycle progression was analyzed by flow cytometry. Cell adhesion and proliferation was determined by CellTiter-Glo luminescent cell viability assay kit. Cell morphological alteration and microtubule stability were analyzed by immunofluorescence staining. Effect of niltubacin, tubacin and NaB on acetylated tubulin and siRNA efficacy were measured by western blotting.ResultsSuppression of histone deacetylase 6 activity significantly compromised the migration and invasion of Burkitt’s lymphoma cells, without affecting cell proliferation and cell cycle progression. Mechanistic study revealed that HDAC6 modulated chemokine induced cell shape elongation and cell adhesion probably through its action on microtubule dynamics.ConclusionsWe identified a critical role of HDAC6 in the metastasis of Burkitt’s lymphoma cells, suggesting that pharmacological inhibition of HDAC6 could be a promising strategy for the management of metastatic Burkitt’s lymphoma.

Inhibition of Activin A Ameliorates Skeletal Muscle Injury and Rescues Contractile Properties by Inducing Efficient Remodeling in Female Mice

Activin A, a member of the transforming growth factor-β superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

Novel Approaches For Targeted Therapy In Cutaneous T-Cell Lymphomas

IntroductionDevelopment of targeted therapy for Cutaneous T-Cell Lymphoma (CTCL) patients still requires actionable mutated genes and deregulated pathways to be identified and explored. Although it has been proposed that neoplastic CTCL cells feature increased TCR downstream signaling and also a degree of phenotypic plasticity, the mechanistic nature of the pathogenesis of this disease remains essentially unveiled. Our laboratory has recently studied the mutational status of a number of human CTCL lesions, and detected TCR/PLCG1 and JAK/STAT signaling pathways frequently mutated. Taking advantage of these findings, we have explored the biological effects that targeted inhibition, using specific inhibitors of the two afore mentioned signaling pathways, exerts in proliferation, survival and phenotype in a panel of human CTCL cell lines. Our results suggest that TCR/PLCG1 and JAK/STAT pathways can control proliferation, survival and phenotype of CTCL cells and hence can serve as potential targets for mono or combinatorial therapy in CTCL patients. Material and MethodsCutaneous T-cell lymphoma cell lines used were HH (MF) and MJ (MF) obtained from ATCC (Rockville, MD, US ) and My-La (MF) and HUT-78 (SS) obtained from ECACC (Salisbury, UK).Cell proliferation analyses were performed using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) with the appropriate amount of inhibitor. Apoptosis were evaluated by flow cytometry using FlowCellect Annexin Red Kit (EMD Millipore Corporation, Billerica, USA). All data were detected on a FACS Calibur flow cytometer (BD) and analyzed using CellQuest Pro software (BD). Treg/Th17 phenotype was determined by flow cytometry. Cells were stained with monoclonal antibodies: anti-CD4-allophycocyanin (APC)-Cy7 (clone SK3, Becton Dickinson, San Diego, CA). Monoclonal antibodies to detect intracellular antigens were: anti-Foxp3-PE (clone PCH101, eBiosciences, San Diego, CA), anti-RORgt-APC (clone AFKJS-9, eBioscience, San Diego, CA), and IgG2a isotype controls conjugated with PE and APC. The cells were acquired by flow cytometer (FACS Canto-II, Becton Dickinson). ResultsTo test both the effect that inhibition of PLCG1 downstream signaling and the effect that inhibition of JAK downstream signaling provokes in CTCL cell lines, they were incubated with increasing concentrations of specific CaN and JAK inhibitors like FK506 and INCB018424 respectively or DMSO (vehicle) for 0, 24 and 48 h. The results show that increasing concentrations of FK506 affected the proliferation and survival of CTCL cells in a concentration dependent manner. But in the case of INCB018424 inhibitor mainly affects CTCL cell proliferation versus survival. The combined use of FK506 and INCB018424 produced a significant greater inhibition of CTCL cell proliferation than each inhibitor alone except of HUT-78, where the combined treatment was not significant (Figure 1). [Display omitted] Because of neoplastic T-cells can acquire Treg and/or Th17 phenotypes at different stages of the disease (Tiemessen, M.M. et al. 2006) and that there is a CTCL phenotypic plasticity(Abraham, Zhang et al. 2011) (Eisenstein and Williams 2009) (Hoechst, Gamrekelashvili et al. 2011), we have decided to study these phenotypes in the four CTCL cell lines. Strikingly we found that most cells in all CTCL cell lines were double positive for the expression of Treg and Th17 markers (Figure 2). [Display omitted] The expression of the Treg and Th17 lineage markers (FoxP3 and RORγt respectively) after treatment with FK506 and INCB018424 were tested. FK506 provoked an unbalance in the expression of the Treg and Th17 markers in all CTCL cell lines. (Figure 3). [Display omitted] ConclusionsThese results are the first evidences for the use of a combinatorial targeted therapy developed on the basis of a mutational analysis in CTCL, which in the future may serve as a lead to explore into detail about a potential utilization of this therapeutical approach in the clinic. Surprisingly, our results evidence a novel hybrid Treg/Th17 phenotype in our panel of CTCL cell lines, that we could alter by interfering specifically with TCR/PLCG1 and/or JAK/STAT downstream signaling using FK506 and/or INCB018424 respectively. Although these are still primary results, it will be important to explore how to take clinical advantage of these phenotypic characteristics in patients. Disclosures:No relevant conflicts of interest to declare.

1674P - Identification of Phosphorylated Ribosomal Protein S6 as a Potential Predictor of Hepatocellular Carcinoma Response to Sorafenib by Pathway-Based Phosphoprotein Profiling

ABSTRACT Backgroud Sorafenib is a multi-kinase inhibitor that has been proved to be effective for the treatment of advanced hepatocellular carcinoma (HCC). However, only a small proportion of patients receiving sorafenib obtain the anticipated therapeutic benefits. It would therefore be desirable to clarify the precise molecular mechanisms that confer resistance to sorafenib and identify a predictive biomarker for unresponsiveness to sorafenib. Methods We constructed reverse-phase protein microarrays (RPPAs) onto which whole lysates of 95 cell lines derived from various tumors, including 23 HCCs, were plotted and examined for phosphorylation expression of 183 proteins serving as nodes in 120 signaling pathways registered in the NCI-Nature curated pathway (NCI-Nature_Curated.bp2.owl 2011.12.19). The sensitivity (IC50 value) of the 23 HCC cell lines to sofafenib was quantified using the CellTiter-Glo Luminescent Cell Viability Assay kit. Results Use of fluorescence dye significantly expanded the sensitivity and dynamic range of the RPPAs. We found that the relative level of phosphorylation of ribosomal protein S6 (p-rpS6) at the Ser235/236 residues showed the highest correlation with sensitivity to sorafenib among the 183 signaling nodes (P = 0.0044, Spearman correlation analysis), followed by phosphorylation of the same protein at the Ser240/244 residues (P = 0.0070). Immunoblot analysis confirmed that the sorafenib-resistant cell lines had the highest level of rpS6 phosphorylation. We found that in some HCC cell lines the mitogen-activated protein kinase (MAPK) pathway was activated downstream of RAF kinase and showed sensitivity to MEK (CI-1040) and RSK (SL0101) inhibitors. Conclusions To achieve personalization of molecular therapies, precise mapping of activated signaling pathways in individual patients is essential. RPPA requires only a small amount of protein and is ideal for application to clinical settings. We are collecting tumor biopsy samples from HCC patients to verify the present findings, and plan to present the data at the meeting. Disclosure All authors have declared no conflicts of interest.

RhoGDI SUMOylation at Lys-138 Increases Its Binding Activity to Rho GTPase and Its Inhibiting Cancer Cell Motility

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Pre-B-Cell Leukemia Transcription Factor 1 Regulates Expression of Valosin-Containing Protein, a Gene Involved in Cancer Growth

Valosin-containing protein (VCP) is involved in a wide variety of cellular functions. Our previous studies showed that the enhanced expression of VCP in cancer cells correlated with invasion and metastasis of cancers. Here, the regulatory mechanism for VCP transcription was investigated. Luciferase reporter constructs containing serially deleted 5'-flanking region of the VCP gene were transfected into MCF7 mammary carcinoma cell line, in which VCP was abundantly expressed. The deletion and mutation at the two binding motifs for pre-B-cell leukemia transcription factor 1 (PBX1) reduced the luciferase activity, indicating that these two PBX1 motifs mediated the transactivation of the VCP gene. Chromatin immunoprecipitation assay showed the binding of PBX-1 to the 5'-flanking region of the VCP gene. The knockdown of PBX1 by siRNA decreased the expression level of VCP. VCP is reported to maintain cell viability after the treatment of tumor necrosis factor-alpha. The viability of tumor necrosis factor-alpha-treated cells was significantly reduced in PBX1 knockdown MCF7. These findings indicate that PBX1 plays a crucial role in VCP expression and function and that the PBX-VCP pathway might be important for cell survival under cytokine stress.