bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B74: Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma
Authors: Moore Nathan F. , Chipumuro Edmond , Hatheway Clark M. , Zhang Tinghu , Gray Nathanael S. , George Rani E.
Published: May 2016

Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma (NB). One of the more common mutations, ALKF1174L, is sensitive to the FDA-approved ALK inhibitor, crizotinib, only at high doses and mediates acquired resistance to crizotinib in ALK-rearranged cancers. To identify compounds that would enhance the cytotoxic effect of crizotinib, we conducted a high throughput small molecule screen for compounds that synergize with crizotinib in NB cells expressing the ALKF1174L mutation. We identified two pan-selective cyclin dependent kinase (CDK) inhibitors, AT7519 and SNS-032, which have overlapping efficacy against the cell cycle-regulating CDK2 and transcriptional elongation-regulating CDK9. Both inhibitors demonstrated synergistic activity with crizotinib, leading to downregulation of pALK and downstream signaling and significantly increased apoptosis over that of either single agent alone. This effect was observed in NB cells expressing not only ALKF1174L, but also in the other commonly observed ALKR1275Q mutation. Synergy was also noted with ceritinib (LDK378), a structurally unrelated ALK inhibitor, in combination with both AT7519 and SNS-032. The combination of crizotinib and SNS-032 led to the inhibition of CDK9-mediated transcriptional elongation as indicated by downregulation of RNA polymerase II phosphorylation at serine 2. Additionally, the combination also induced proteolytic cleavage of elongation-regulating BRD4, suggesting alternative mechanisms contributing to transcriptional inhibition in addition to direct inhibition of CDK9 activity These findings were comparable with results obtained using compounds that were highly selective for CDK7, which has roles in transcriptional initiation as well as in CDK9 activation. Finally, in murine xenogaft models of ALK-mutated NB, the combination led to inhibition of tumor growth and prolongation of survival compared to single agents alone. Together, these data support further pre-clinical and clinical efforts to explore the therapeutic potential of combining ALK inhibitors with transcriptional CDK inhibitors in ALK-mutated NB.

Citation Format: Nathan F. Moore, Edmond Chipumuro, Clark M. Hatheway, Tinghu Zhang, Nathanael S. Gray, Rani E. George. Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B74.
Abstract B80: New inhibitors of histone deacetylase 8 (HDAC8) as anticancer agents
Authors: Jung Manfred , Chakrabarti Alokta , Heimbach Tino , Sippl Wolfgang , Marek Martin , Romier Christophe , Oehme Ina , Witt Olaf
Published: May 2016

The protein deacetylase HDAC8 has been recognized to be a potential target for the treatment of certain types of cancer, e.g. CNS cancer or T-cell leukemia. Starting out froom HDAC8 inhibitors with potency in the low micromolar region we have prepared a series of analogues and subjected them to iterative cycles of in-vitro testing using a fluorescence based assay and subsequent synthetic variation. Molecular docking to available X-ray structures of human HDAC8 further supported the process. We have obtained potent HDAC8 inhibitors in-vitro (potency down to 26 nM) and high selectivity over hHDAC1 and 6(>200fold). Selected inhibitors block the proliferation of different cancer cell lines in culture (SH-SY5Y glioblastoma, Jurkat T-cell leukemia) and show hyperacetylation (shown by Western blotting) of the HDAC8 target Ac-SMC3.

Thus, we have obtained potent and selective HDAC8 inhibitors with cellular activity and target engagement. Mechanistic studies are underway to further optimized selected compounds.

Citation Format: Manfred Jung, Alokta Chakrabarti, Tino Heimbach, Wolfgang Sippl, Martin Marek, Christophe Romier, Ina Oehme, Olaf Witt. New inhibitors of histone deacetylase 8 (HDAC8) as anticancer agents. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B80.
Abstract B86: Impact of multi targeted epigenetic therapy (MTET): a series of 100 consecutive advanced solid tumor cancers
Authors: Nezami M. A. , Hager Steven , Lanman Richard
Published: May 2016

Background:

Tumor heterogeneity is a key underlying factor that leads to resistance to chemo-and matched therapies. Circulating cell-free tumor DNA (ctDNA) may capture more tumor heterogeneity than a tissue biopsy-derived DNA analysis. In addition, markers of vasculogenesis have been shown to be sensitive markers of tumor growth vs. response to treatment. Here we study the impact of a novel multitargeted epigenetic therapy (MTET) on ctDNA, markers of vasculogenesis and radiographic response.

Methods:

One hundred consecutive patients with advanced solid tumor cancers were treated with multi targeted epigenetic therapy (MTET). More than 60% were stage IV, with 52% male and 48% women, 24 to 81 years of age. Pre-treatment and post-treatment imaging, circulatory tumour cells (CTC), (Biofocus Lab), ctDNA (Guardant Health, Inc.) and plasma VEGF (LabCorp) were obtained. PET scan metabolic activities were determined by a radiologist in a tertiary hospital based metabolic imaging canter. The average patient had undergone two or more lines of therapy previously. Patients underwent 10 or more cycles of a novel multitargeted epigenetic therapy (MTET), consisting the administration of polyphenols intravenously, either independently or in addition to conventional chemotherapies, consisting of standard and palliative chemotherapies.

Results:

Overall positive imaging response rate(in favor for metabolic response) was observed in 62%. The magnitude of response was independent of type of tumor. Among the patients who had circulating tumor cells (CTC) and/or ctDNA analyzed(number 20 and number 4 respectedly), a positive response (80% for CTC) was seen with improved CTCs and/or the ctDNA alterations for the gene variant at highest mutant allele. Reduction and normalization of plasma VEGF were seen in 92% of patients. Tolerability was good and none of the patients discontinued therapy due to toxicity.

Conclusions:

There were significant radiographic responses in advanced solid tumor cancer patients receiving MTET therapy. These changes were paralleled by the changes in serological assessment of vasculogenic factors and/or CTC/ctDNA. The high response rate is unusual for patients who have undergone multiple lines of therapy and a prospective randomized therapy of MTET is being planned.

Citation Format: M. A. Nezami, Steven Hager, Richard Lanman. Impact of multi targeted epigenetic therapy (MTET): a series of 100 consecutive advanced solid tumor cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B86.
Abstract B89: Mechanistic assessment of heat shock network perturbation by novel chemotypes identified using chemogenomic screening
Authors: Goode Kourtney M. , Thomas Fiona M. , Pascuzzi Pete , Davisson V. Jo , Hazbun Tony R.
Published: May 2016

The 90-kDa heat shock protein 90 (Hsp90) is a chaperone protein responsible for the folding and activation of numerous proteins disregulated in various cancers that drive oncogenesis. Consequently, inhibition of Hsp90 is thought to be an attractive anti-cancer therapeutic strategy, though to date, clinical success has been limited. Hsp90 inhibition leads to simultaneous degradation of multiple oncogenic client proteins, giving “polypharmacologic” effects through the targeting of a single, highly networked protein. Hence, the development of small molecule inhibitors of Hsp90 may be effective anti-cancer agents that have reduced likelihood of development of resistance when compared to other less networked single protein targets. This study demonstrates identification and validation of a novel chemotype that perturbs the heat shock protein network. A liquid culture-based phenotypic screen employing Saccharomyces cerevisiae haploid deletions strains with known sensitivities to Hsp90 inhibitors by screening of the NCI diversity II chemical library set. The screen identified 9 hit compounds; 2 previously identified heat shock modulators and 7 novel heat shock modulator hits. To validate these novel compounds as heat shock network selective, we bioinformatically show that the NSC145366 compounds sensitivity profile in NCI tumor cell lines significantly correlates with other Hsp90 inhibitors. Also, biochemical analyses demonstrate NSC145366 has direct interactions with two cytoplasmic isoforms of human Hsp90 both in recombinant protein preparations and in crude tumor cell lysates. In addition, we show that the compound preferentially interacts with the Hsp90 C-terminal dimerization domain as opposed to many of the current N-terminal domain-based inhibitors. Further characterization of the compound interaction with Hsp90 and its effects on the heat shock cellular network are ongoing. This study demonstrates the utility of phenotypic screening to identify heat shock protein network modulators and the possibility inhibiting Hsp90 in a novel and different mode from previous inhibitors.

Citation Format: Kourtney M. Goode, Fiona M. Thomas, Pete Pascuzzi, V. Jo Davisson, Tony R. Hazbun. Mechanistic assessment of heat shock network perturbation by novel chemotypes identified using chemogenomic screening. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B89.
Abstract C127: Chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC activities
Authors: Kato Yukinari , Kaneko Mika K.
Published: May 2016

Background: Podoplanin (PDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in a variety of tumors, and binds to C-type lectin-like receptor-2 (CLEC-2). Overexpression of PDPN is involved in invasion and metastasis. Several anti-PDPN monoclonal antibodies (mAbs) such as NZ-1 showed antimetastatic activities by binding to platelet aggregation-stimulating (PLAG) domain of PDPN. Recently, we developed a novel mouse anti-PDPN mAb, LpMab-7 using the cancer-specific mAb (CasMab) method. LpMab-7 is more sensitive than anti-PLAG domain mAbs. Methods: We generated and characterized a mouse-human chimeric anti-PDPN mAb, chLpMab-7. We investigated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities in vitro. Furthermore, we evaluated the in vivo efficacy of chLpMab-7 using both xenograft models and experimental metastasis models. In xenograft models, PDPN-expressing CHO (CHO/hPDPN) or a lung cancer cell line PC-10 was engrafted into nude mice. In experimental metastasis models, CHO/hPDPN cells were inoculated into tail vein of the nude mice. Results: ChLpMab-7 antibody showed ADCC and CDC activities against CHO/hPDPN, glioblastoma, mesothelioma, or lung cancer cell lines. Treatment with chLpMab-7 abolished both tumor growth in xenograft and spontaneous pulmonary metastasis. ChLpMab-7 exhibited dramatic suppression of experimental pulmonary metastasis, even when it was injected five days after the inoculation of tumor cells. Conclusion: ChLpMab-7 suppressed tumor development and hematogenous metastasis via ADCC/CDC activities. PDPN should be useful as a novel antibody-based therapy.

Citation Format: Yukinari Kato, Mika K. Kaneko. Chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC activities. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C127.
Abstract C131: ‘KRAS addiction’ an artifact of 2D culture? Inhibitors of the mut-KRAS NSCLC 3D growth
Authors: Kirkpatrick D. Lynn , Delaney Roisin , Grandjean Geoff , Madrigal Assael , Indarte Martin , Scott Mike , Powis Garth
Published: May 2016

Introduction: KRAS, the predominant form of mutated RAS (mut-KRAS), is found in ∼25% of patient tumors across many cancer types and plays a critical role in driving tumor growth and resistance to therapy. We identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) to be critical for mut-KRAS but not wild type (wt)-KRAS signaling and cell proliferation. Its product CNK1 is a multi-domain organizer protein found as part of the Ras membrane signaling nanocluster where it binds to mut-KRas although not to wt-KRas, and is necessary for mut-KRas cell growth and signaling. We have exploited the pleckstrin homology (PH)-domain of CNK1 as a target for drug discovery to inhibit mut-KRas. To understand the role of CNK1 as a regulator of KRas cell growth, evaluation of siKRAS and siCNK1 on 2D and 3D growth were evaluated. We also studied the effects of KRAS cell growth conditions on response to selective inhibitors the PH-domain of CNK1 identified using a computational modeling approach.

Results: Using a panel of twelve NSCLC lines and siRNA knockdown of KRAS we found that the reported growth addiction of some cancer cell lines for mut-KRAS, and the resistance of others, is most likely an artifact of 2D culture. Mut-KRAS NSCLC cell lines showing addiction in 2D growth did not show addiction in 3D anchorage independent growth in agarose, where all cell lines were more sensitive. Wt-KRAS cell lines were largely unaffected by siKRAS knockdown. Additionally, the CNK1 inhibitor PHT-7390 IC50s for 2D growth inhibition of a panel of 8 mut-KRAS NSCLC lines ranged from 0.60 to 100 μM (ave 18.7 μM) yet was found to be considerably more potent in 3D culture with IC50s from 0.03 to 2.99 μM (ave 0.69 μM). The most pronounced difference was seen in H2009 and Calu1 mut-KRAS cells where PHT-7390 IC50s in 2D were >100 μM, while 0.093 and 0.35 μM in 3D. Wt-KRas NSCLC growth inhibition (3 lines) was largely unaffected by PHT-7390 under either condition with average IC50s of 68 and 69 μM in 2D and 3D conditions, respectively. Target engagement in 2D has shown these CNK1 inhibitors block mut-KRAS signaling but not that of wt-KRAS.

Conclusion: Variable enhanced growth dependence on mut-KRAS (“addiction”) is seen in 2D but not 3D. Potent and specific inhibition of mut-KRAS cell line growth by CNK1 PH-domain inhibitors is considerably more robust in 3D culture suggesting a novel approach to inhibit mut-KRAS effect on cancer growth.

Citation Format: D. Lynn Kirkpatrick, Roisin Delaney, Geoff Grandjean, Assael Madrigal, Martin Indarte, Mike Scott, Garth Powis. ‘KRAS addiction’ an artifact of 2D culture? Inhibitors of the mut-KRAS NSCLC 3D growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C131.
Abstract C142: Functional characterization of P58IPK-PERK pathway in prostate cancer cells
Authors: Sheng Xia , Storm Margrethe , Jin Yang , Saatcioglu Fahri
Published: May 2016

Androgen signaling is important for the normal development and function of the prostate as well as for prostate cancer (PCa). We recently found that androgens activated the IRE1α-XBP1 arm of the canonical unfolded protein response (UPR) pathways and simultaneously inhibited PERK-eIF2α signaling. Activation of the IRE1α-XBP1 arm was mediated by direct binding of the androgen receptor (AR) in the vicinity of IRE1, as well as XBP1s target genes, and increase in their expression. Consistently, AR and IRE1α pathway gene expression are correlated in human PCa samples and XBP1s protein expression is significantly increased in cancer compared to normal prostate. However, the mechanisms behind androgen mediated inhibition of PERK-eIF2α signaling are not clear at present. One possible mediator in this regard is P58IPK, an XBP1s target gene that can interact with and inhibit PERK and eIF2α phosphorylation. Here, we show that P58IPK expression is increased by androgens in a time-dependent manner and that it plays a pro-survival role in PCa cells. P58IPK knockdown activated PERK expression as well as subsequent eIF2α phosphorylation when induced with the UPR activator thapsigargin. These findings suggest that P58IPK mediates, at least in part, the differential androgen effects between the IRE1α and PERK signaling in PCa and may be a potential therapeutic target.

Citation Format: Xia Sheng, Margrethe Storm, Yang Jin, Fahri Saatcioglu. Functional characterization of P58IPK-PERK pathway in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C142.
Abstract C155: Cyclin-dependent kinase 1 (Cdk1) is a promising therapeutic target to overcome ovarian cancer
Authors: Cho Hanbyoul , Yang Wookyeom , Shin Ha-Yeon , Lee Eun-ju , Chay Doo-Byung , Kim Jae-Hoon
Published: May 2016

Purpose: Ovarian cancer shows heterogeneous characteristics which cause chemoresistance and recurrence. The goal of this study was to elucidate characteristics common to various ovarian cancers in order to evaluate their potential for targeted therapy.

Methods: Microarray results for cell lines of serous, mucinous, and brenner types of ovarian cancers were reanalyzed and validated via ovarian cancer cell lines, GEO datasets and TMA blocks. RO-3306 which is an inhibitor of Cdk1 and si-cdk1 was used to measure the growth rate of ovarian cancer cell lines via FACS analysis. In addition, combination treatment with RO-3306 and cisplatin was administered in vitro and in in vivo xenograft mouse model.

Results: Microarray reanalysis revealed that expression of Cdk1 and cyclinB1 was higher in 5 types of ovarian cancer cell lines, 4 GEO datasets and ovarian cancer TMA blocks than in HOSE cells. Ovarian cancer TMA particularly showed increased level of cytoplasmic Cdk1 protein, but not in nucleus. When Cdk1 was inhibited by siRNA and a potent Cdk1 inhibitor (RO-3306), growth of ovarian cancer cells decreased, while G2/M arrest or apoptosis increased. In addition, tumor growth was significantly lesser in a xenograft mouse model treated with a combination of RO-3306 and cisplatin than in mice treated with each drug alone.

Conclusions: Cdk1 is a promising gene for targeted anticancer therapy. It is expected that combined treatment with Cdk1 inhibitor and chemotherapeutic agents would maximize the effects of ovarian cancer treatment.

Citation Format: Hanbyoul Cho, Wookyeom Yang, Ha-Yeon Shin, Eun-ju Lee, Doo-Byung Chay, Jae-Hoon Kim. Cyclin-dependent kinase 1 (Cdk1) is a promising therapeutic target to overcome ovarian cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C155.
Abstract C31: Vemurafenib selectively radiosensitizes BRAF V600E mutant papillary and anaplastic thyroid carcinoma cells in vitro
Authors: Robb Ryan N. , Yang Linlin , Chatterjee Moumita , Saji Moto , Ringel Matt , Chakravarti Arnab , Williams Terence
Published: May 2016

Background: BRAF mutations are oncogenic drivers which occur in about 60% of papillary thyroid cancer (PTC) as well as a significant proportion of anaplastic thyroid cancer (ATC). BRAF mutations drive MAPK signaling and treatment resistance in thyroid cancer. BRAFV600E is the most common of these mutations in PTC, and can be selectively inhibited by vemurafenib.

Methods: We used a panel of PTC and ATC cell lines to assess how the presence/absence of BRAFV600E impacts radiation sensitivity. We used radiation clonogenics, comet assays, nuclear foci formation, and western blots to determine the effect of vemurafenib on PTC and ATC cell lines.

Results: Analysis of radiation clonogenics revealed relative radioresistance in cell lines containing the BRAFV600E mutation versus wild-type. Vemurafenib inhibited MAPK signaling in V600E mutants, but showed no effect on BRAF wild-type cell lines. Vemurafenib pretreatment selectively radiosensitized BRAFV600E mutants, as assessed by radiation clonogenic assays. Neutral comet assays also showed that vemurafenib impairs DNA repair in BRAFV600E lines. Furthermore, γ-H2A.x westerns and nuclear foci staining indicated that vemurafenib pretreatment decreases the ability of cells to repair double-strand DNA breaks in cell lines containing the BRAFV600E mutation. Vemurafenib also appeared to alter the kinetics of nuclear foci formation and resolution of 53BP1 as well as Rad51 in these cell lines.

Conclusions: From our initial results, BRAF mutations appear to be associated with radioresistance in PTC and ATC cell lines. Vemurafenib selectively radiosensitizes both PTC and ATC cells through inhibition of DNA repair mechanisms. Together, these data suggest that combining vemurafenib and radiation may improve therapeutic control for BRAFV600E mutant thyroid cancers.

Citation Format: Ryan N. Robb, Linlin Yang, Moumita Chatterjee, Moto Saji, Matt Ringel, Arnab Chakravarti, Terence Williams. Vemurafenib selectively radiosensitizes BRAF V600E mutant papillary and anaplastic thyroid carcinoma cells in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C31.
Abstract C40: Inhibition of Mcl-1 through covalent modification of a non-catalytic lysine side chain
Authors: Su Qibin , Akçay Gizem , Grimster Neil , Belmonte Matthew , Rawlins Philip , Lamb Michelle , Hird Alexander , Aquila Brian
Published: May 2016

Myeloid cell leukemia 1 (Mcl-1), a potent anti-apoptotic protein of the BCL2 family, has been studied as a key resistance factor in human cancers. Restoring apoptotic signals by inactivating Mcl-1 protein interactions with small molecule inhibitors has been intensively pursued as targets for cancer therapeutics. We herein describe our effort towards the structure-based design, synthesis and evaluation of first potent, covalent binders of Mcl-1. The resulting inhibitors specifically modified a non-catalytic lysine residue with high level of Mcl-1 potency in biochemical and cell based assays. Our covalent inhibitors could provide potent probes to interrogate Mcl-1 dependent cancer biology.

Citation Format: Qibin Su, Gizem Akçay, Neil Grimster, Matthew Belmonte, Philip Rawlins, Michelle Lamb, Alexander Hird, Brian Aquila. Inhibition of Mcl-1 through covalent modification of a non-catalytic lysine side chain. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C40.
Abstract C66: SPHK1 expression associated with acquired resistance to oxaliplatin in colon cancer cell lines
Authors: Lee Hee Yeon , Lee Myung Ah , Park Jin Hee
Published: May 2016

Background Oxaliplatin is a main chemotherapeutic agent in the treatment of advanced colorectal cancer. The response rate has been reported to be over 50%, especially when combined with a target agent. However, most patients develop resistance to oxaliplatin even in responding patients, leading to cancer related death due to organ dysfunction. Unfortunately the mechanism of resistance of oxaliplatin is unclear. In the present study, we tried to identify the role of Sphingosine Kinase 1 (SPHK1) involved in the acquired resistance to oxaliplatin using colon cancer cell line.

Method We treated HCT-116 colon cancer cell lines with gradient concentrations of oxaliplatin starting at the dose of IC50. After treated with initial dose of oxaliplatin for 48hrs, we collected the viable cell and then treated again with elevated dose of oxaliplatin. After prolonged period of exposure to low dose of oxaliplatin, we compared cell viable test and protein expression between HCT-116 wild cell and oxaliplatin treated HCT-116 cell line. We also investigated the change of response to oxaliplatin after tranfection of SPHK1 into wild type of HCT-116 cell line. The viability was determined by Neutral Red uptake assay.

Result After prolonged repeated exposure of low dose oxaliplatin, HCT-116 cell line showed resistance to oxaliplatin and expressed new protein. We identified this new protein as SPHK1 using protemics. Comparing the wild type and the resistance cell line, phosphorylated Akt, phosphorylated ERK, and PTEN expression were upregulated in the resistant cell line. After transfection of SPHK1 into the wild type HCT-116 cell line, response to oxaliplatin decreased comparing to the control group.

Conclusion Based on our data, SPH1K can be a potential factor for the acquired resistance to oxaliplatin in colon cancer. It is not known yet the association between SPHK1 and PTEN in the mechanism of resistance. Currently, we are conducting experiment to identify these associations and mechanism of acquired resistance using other cell lines and human tissue.

Citation Format: Hee Yeon Lee, Myung Ah Lee, Jin Hee Park. SPHK1 expression associated with acquired resistance to oxaliplatin in colon cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C66.
Abstract C76: A novel small molecule inhibitor of constitutive androstane receptor (CAR) that resensitizes doxorubicin-resistant neuroblastoma cells
Authors: Cherian Milu T. , Takwi Apana A. , Lin Wenwei , Chen Taosheng
Published: May 2016

This study aims to identify new specific small molecule inhibitors of the xenobiotic receptor CAR (constitutive androstane receptor) which could be used to increase sensitivity of resistant cancer cells to chemotherapeutics.

CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR thus rendering them mechanistically counterproductive in tissues expressing both receptors. Using a directed screening approach, we discovered a potent small molecule, CINPA1, capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM, without activating PXR. CINPA1 is a specific xenobiotic inhibitor and has no general cytotoxic effects up to 25 μM. CINPA1 effectively inhibits CAR-mediated gene expression in all donors of human hepatocytes that express CAR endogenously. The mechanisms by which CINPA1 inhibits CAR function were extensively examined. We show that CINPA1 association with the CAR ligand binding domain results in increased corepressor interaction and reduced recruitment of coactivators. In addition, chromatin immunoprecipitation in the presence of CINPA1 illustrates reduced CAR binding to the promoter regions of target genes.

Multidrug resistance in tumors after chemotherapy can be associated with errant CAR activity, as in the case of neuroblastoma. Treatment of doxorubicin-resistant neuroblastoma cells with CINPA1 resulted in reduced CAR activity and subsequent decrease in drug-transporter levels. In doxorubicin-resistant UKF-NB3 (neuroblastoma) cells, CINPA1 treatment increased doxorubicin sensitivity and decreased cell growth. This suggests that CAR inhibitors used in combination with existing chemotherapeutics could potentially be utilized to attenuate multidrug resistance and resensitize chemo-resistant cancer cells.

Citation Format: Milu T. Cherian, Apana A. Takwi, Wenwei Lin, Taosheng Chen. A novel small molecule inhibitor of constitutive androstane receptor (CAR) that resensitizes doxorubicin-resistant neuroblastoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C76.
Abstract C91: Reversible lysine specific demethylase-1 inhibition is synergistic with docetaxel in castrate refractory prostate cancer
Authors: Gupta Sumati , Weston Alexis , Bearss Jared , Sharma Sunil
Published: May 2016

Lysine specific demethylase-1 (LSD-1) expression correlates with poor survival in prostate cancer. LSD-1 is also key in androgen receptor signaling in androgen sensitive and castration refractory prostate cancer. LSD-1 is a chromatin modifying enzyme that in conjunction with its binding partners functions as an ‘erasor’ in the Epigenetic code process and demethylates lysine 4 and 9 residues on H3 protein resulting in gene repression or activation. Specifically in prostate cancer it demethylates H3k9me2 playing an important role in androgen receptor signaling promoting myc expression. Docetaxel chemotherapy is effective in prostate cancer but limited by toxicity and development of resistance. In this study we demonstrate synergy between LSD-1 inhibition and docetaxel in castrate refractory cell lines. We further demonstrate that LSD-1 inhibition is effective in docetaxel resistant cell lines. Cell survival assays were performed with HCI2509 on castrate refractory and docetaxel resistant prostate cancer cell lines. Histone methylation and myc expression were assessed. Colony formation assays, cell cycle analysis, xenograft studies were performed. Synergy studies were performed to assess the combination of 2509 and docetaxel. HCI2509 is cytotoxic to castrate refractory and docetaxel resistant prostate cancer cell lines in low micromolar doses. Treatment with HCI2509 resulted in dose dependent increase in H3K4me2, H3K4me3, H3K9me2 levels and decrease in myc protein. RT-PCR showed a significant decrease in MYC expression upon treatment with HCI2509. Cell cycle progression was arrested at G0/G1 upon treatment with 2509 with inhibition of colony formation at 10 to 100 fold lower concentrations. PC3 xenografts in mice showed significant reduction in tumor burden upon treatment with 2509 with increased H3K9me2 marks and reduced myc protein levels. LSD-1 inhibition with HCI2509 has therapeutic potential in castrate refractory and docetaxel resistant prostate cancer. It inhibits myc expression and sensitizes castrate refractory prostate cancer to docetaxel.

Citation Format: Sumati Gupta, Alexis Weston, Jared Bearss, Sunil Sharma. Reversible lysine specific demethylase-1 inhibition is synergistic with docetaxel in castrate refractory prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C91.
Abstract CN03-01: Toxicities from immunomodulatory antibodies: An overview
Authors: Italiano Antoine
Published: May 2016

immunomodulatory monoclonal antibodies used to strengthen the immune system against cancer have shown remarkable efficacy for patients with advanced melanoma and non-small cell lung cancer and are likely to significantly improve the survival of patients with several other malignancies.

Events of clinical interest of a potential immunologic etiology (irECIs) may be defined as an adverse event of unknown etiology, associated with drug exposure and consistent with an immune phenomenon. Immune related adverse events (irAEs) may be predicted based on the nature of the compound and its mechanism of action. An irAE can occur shortly after the first dose or several months after the last dose of treatment. Such irAE represent a unique spectrum of side effects including dermatologic, gastrointestinal, hepatic, endocrine, and other less common inflammatory events. Even if most of them are mild and transient, they can be ocasionally severe or fatal. We will review the specific side effects of antibodies targeting the PD-1/PDL1, CTLA-4 and CSF1 receptors and discuss their physiopathology and potential impact on patient management.

Citation Format: Antoine Italiano. Toxicities from immunomodulatory antibodies: An overview. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN03-01.
Abstract CN05-04: Serial killing of tumor cells by cytotoxic T cells: Mechanisms and implications for therapy
Authors: Friedl Peter
Published: May 2016

Cytotoxic T lymphocytes (CTL) eliminate tumor target cells in an antigen and cell-contact dependent manner, both spontaneously and when activated by immunotherapy. Lethal hit delivery is considered to be a rapid and binary “yes/no” process under conditions of high immunogenicity. In tissues, killing efficacy further depends upon sequential conjugation of CTL with multiple target cells, termed “serial killing.” Here we show that elimination of cancer cells results from a cooperative process executed by multiple CTL engaging sequentially with the same target cell. Migrating CTL transit between target cells and cooperate to initiate apoptosis by a series of sublethal interactions (additive cytotoxicity), whereas individual conjugations rarely reached thresholds to induce apoptosis. Consequently, in invading B16F10 melanoma tumors treated with adoptive T cell therapy in vivo, serial engagements and tumor-cell apoptosis induction were confined to regions with high CTL density, which supported CTL accumulation and additive cytotoxicity. Highest rates of serial engagements followed by tumor eradication were reached in the invasive tumor front where moving tumor cells and CTL collided within the same pro-migratory tissue compartment, suggesting invading tumor cells as target for immunotherapy. Thus, additive cytotoxicity requires CTL cooperation and serial engagements with target cells to deliver cumulative sublethal events and successfully eradicate solid tumor cells. The need for additive “hits” has implications for topographic mechanisms of immune evasion of tumor cells as well as immune intervention to enhance CTL accumulation and cooperation.

Citation Format: Peter Friedl. Serial killing of tumor cells by cytotoxic T cells: Mechanisms and implications for therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN05-04.
Abstract ED01-02: Gene-modified T cells as biotherapeutics for cancer
Authors: Gottschalk Stephen
Published: May 2016

Adoptive immunotherapy of T cells may have significant advantages over alternative immunotherapeutic approaches such as cancer vaccines since the phenotype, activity and specificity of expanded cells can be analyzed prior to injection. T cells can be genetically engineered prior to infusion to modify the antigens they target, increase their potency, and reduce their susceptibility to tumor-mediated immune inhibition. Genetic modification also allows T cells to be tracked in vivo to learn their fate. Adoptive transfer of T cells has been most successful after hematopoietic stem cell transplantation (HSCT) as prophylaxis and therapy for viral associated disease. In addition, the adoptive transfer of tumor infiltrating lymphocytes (TILs) in combination with lymphodepleting chemotherapy has shown promising long-term antitumor effects in patients with melanoma. Lastly, the adoptive transfer of T cells that have been genetically engineered to be tumor specific has resulted in impressive clinical responses, especially in patients with hematological malignancies. Despite these successes, clinical studies have also highlighted current limitations of adoptively transferred immune cells, including limited expansion in vivo, lack of homing to tumor sites, and toxicities due to recognition of normal tissues. In my talk I will review the current status of T-cell therapy for cancer with special focus on solid tumors and genetically modified T cells.

Citation Format: Stephen Gottschalk. Gene-modified T cells as biotherapeutics for cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr ED01-02.
Abstract LB-A20: Novel Dual Oncogenic Target Inhibitor against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3-Kinase (PI3K)
Authors: Ross Brian D. , Hong Hao , Wang Hanxiao , A, Nino Charles , Van Dort Marcian E.
Published: May 2016

The Ras/MEK/ERK and PI3K/Akt/mTor pathways play a central role in the regulation of normal cell growth, division and differentiation. Dysregulation of these signaling pathways driven by oncogenic mutations/activation leading to elevated kinase activity has been demonstrated in many human cancers. Strong evidence suggests the existence of a feedback loop with crosstalk between these two signaling cascades leading to redundancy in survival pathways. Consequently, monotherapy targeting a single cascade may be insufficient to induce tumor cell death due to drug resistance mechanisms. Initial biological results are presented from a series of novel small molecule kinase inhibitors specifically designed to simultaneously target both MEK1 and PI3K. Structural analogs of the ATP-competitive PI3K inhibitor ZSTK474 and the ATP-noncompetitive class of MEK inhibitors PD0325901, respectively, were covalently combined to provide single compound dual inhibitors. Inhibitors showed potent MEK1 inhibition (0.015 < IC50 (nM) < 56.7) and PI3K inhibition (54 < IC50 (nM) < 341) in tumor cell culture inhibition assays. Cellular efficacy studies were accomplished and revealed several of the designed inhibitor analogs demonstrated near complete inhibition of MEK1 and PI3K kinase activity by western blot analysis in A549 lung adenocarcinoma and D54 glioma cell lines at concentrations as low as 5 μM. Inhibitors also produced dose-dependent decreases in cell viability in these cell lines similar to that displayed with the combined administration of equivalent doses of ZSTK474 and PD0325901. In vivo evaluation of selected compounds was undertaken to evaluate oral bioavailability and activity. In preliminary studies conducted in D54 glioma tumor bearing mice, a 95% and 67% inhibition of tumor ERK1/2 and Akt phosphorylation, respectively, was found at 2 h post-administration as determined by western blot analysis. This study reveals that dual targeted small molecules designed to inhibit MEK1/PI3K pathways were effective in target modulation in intact mouse tumor models. Overall, this study details a highly novel approach for combined MEK1/PI3K inhibition with first in vivo results.

Citation Format: Brian D. Ross, Hao Hong, Hanxiao Wang, Charles A, Nino, Marcian E. Van Dort. Novel Dual Oncogenic Target Inhibitor against Allosteric Mitogen-Activated Protein Kinase (MEK1) and Phosphatidylinositol 3-Kinase (PI3K). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A20.
Abstract LB-A25: PTEN loss promotes phosphorylation-dependent acetylation and inhibition of SPOP tumor suppressor
Authors: An Jian , Huang Haojie
Published: May 2016

The Cullin 3-based E3 ubiquitin ligase substrate-binding protein SPOP is inactivated in approximately 8-15% of human prostate cancers due to gene mutation. Intriguingly, SPOP mutations and inactivation of the PTEN tumor suppressor in human prostate cancers are mutually exclusive. However, it is unclear whether SPOP remains functionally active in PTEN-deficient prostate cancer cells. In the current study, we demonstrate that PTEN loss induces phosphorylation and nuclear exportation of SPOP. We identify a putative cyclin-dependent kinase (CDK) phosphorylation site (serine 222) in SPOP BTB domain, a motif interacting with Cullin 3 and demonstrate that PTEN loss induces SPOP cytoplasm accumulation in a manner dependent on CDK2-mediated phosphorylation of SPOP at serine 222. We further show that serine 222 phosphorylation impairs dimerization and the E3 ligase activity of SPOP without affecting its interaction with Cullin 3. We also identify a potential p300 acetylation site in the C-terminal nuclear localization sequence of SPOP. We demonstrate that acetylation by p300 promotes SPOP translocation from nucleus to cytoplasm and this effect is enhanced by CDK2-mediated phosphorylation of SPOP at serine 222. In contrast, Sirt 2 and Sirt 6 of the sirtuin family are verified to be responsible for deacetylation of SPOP. In the conditional Pten knockout prostate cancer mouse model, Pten deletion induces cytosolic accumulation of SPOP, but the process is reversed in Pten and p300 double knockout prostatic cells. Collectively, our findings reveal cytoplasmic localization and enzymatic inactivation of SPOP as a previously uncharacterized mechanism that contributes to PTEN loss-induced prostate tumorigenesis. They also suggest that blocking SPOP cytoplasm localization represents a viable therapeutic option for treatment of prostate cancers harboring PTEN inactivation.

Citation Format: Jian An, Haojie Huang. PTEN loss promotes phosphorylation-dependent acetylation and inhibition of SPOP tumor suppressor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A25.
Abstract LB-B03: miR-24 acts as a tumor suppressor and radiosensitizer by targeting Jab1/CSN5 functions
Authors: Claret Francois X. , Wang Sumei , Pan Yunbao , Yang Huiling
Published: May 2016

Radiotherapy is the standard therapy for head and neck cancer; however, radioresistance can hinder successful treatment and is likely to give rise to local recurrence and distant metastatic relapse. Recent studies revealed the role of microRNA (miRNA)-mRNA mediated regulation in tumorigenesis; however, whether specific miRNAs regulate tumor radioresistance and can be exploited as radiosensitizing agents remains unclear. The purpose of this study was to identify an miRNA signature from differentially expressed miRNAs in radioresistance. Here, we report that miR-24 acts as a tumor suppressor and radiosensitizer in nasopharyngeal carcinoma (NPC) cells and mouse models by targeting Jab1/CSN5 and that loss of miR-24 is highly associated with poor survival. When combined with irradiation, miR-24 acted as a radiosensitizer in NPC cells. Jab1/CSN5 functioned in a manner opposite that of miR-24 in NPC tumorigenesis and radioresistance. We demonstrated that miR-24 inhibits Jab1/CSN5 translation, leading to tumor growth inhibition, and sensitizes NPC tumors to radiation in vivo. Moreover, miR-24 inhibits DNA damage repair by targeting JAB1/CSN5. Finally, comparative analysis of paired samples of primary and matched recurrent NPC tissues showed that miR-24 levels were significantly lower and Jab1/CSN5 levels were higher in recurrent NPC than in primary NPC. Our findings identify miR-24 as a tumor suppressor and radiosensitizer miRNA and reveal a new therapeutic strategy for radioresistant tumors.

Citation Format: Francois X. Claret, Sumei Wang, Yunbao Pan, Huiling Yang. miR-24 acts as a tumor suppressor and radiosensitizer by targeting Jab1/CSN5 functions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B03.
Abstract LB-B08: Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting
Authors: Tseng Hubert , Amato Robert J. , Brobey Reynolds , Ionescu-Zanetti Cristian , Jensen Jeff , Gage Jacob , Desai Pujan , Liao Angela , Dehghani Mehdi , Rosenblatt Kevin , Souza Glauco
Published: May 2016

Background - A challenge in the analysis of circulating tumor cells (CTCs) is their scarcity and the inability to expand them for further analysis. To overcome this obstacle, we used magnetic 3D bioprinting to form CTC spheroids that could grow. The principle of magnetic 3D bioprinting is the magnetization of cells with nanoparticles and their subsequent printing into spheroids. For this project, CTCs were aggregated into close contact to facilitate interactions and growth in culture. We then demonstrated the ability to perform next generation sequencing (NGS) of the spheroids to detect somatic mutations from renal and prostate cancers.

Methods - Blood samples from prostate and kidney cancer patients were enriched for CTCs (Isoflux, Fluxion Biosciences), from a starting blood volume of 7.5-14 mL. CTCs were isolated immunomagnetically for EpCAM+ EGFR+ cells, then enumerated for CK+ CD45-. The cells were then magnetized by incubation with NanoShuttle (NS, Nano3D Biosciences) and printed into spheroids in 384-well plates. After 4 d of growth, the cells were lysed and DNA was amplified by whole genome amplification (WGA) with the NGA kit (Fluxion Biosciences) and quantified via qPCR. Targeted libraries were sequenced using the PGM (ThermoFisher) sequencing instrument; data was analyzed using a customized variant calling/filtering pipeline based on standard Ion Reporter alignment tools and VarSeqTM for variant filtering and functional interpretation.

Results - CTCs were successfully aggregated using magnetic 3D bioprinting and grew over 4 d. For both prostate and renal cancers, we then demonstrated the detection of somatic variants within a majority of the samples. Using the commercial Oncomine® test kits (ThermoFisher), we found a median of 5 COSMIC variants (32 total) per sample using cell cultures.

Conclusions - We successfully developed a method to aggregate CTCs using magnetic 3D bioprinting, expanded them, and then identified somatic mutations using NGS. This procedure may form the basis of a liquid biopsy-derived molecular testing platform for monitoring urological tumor progression and planning treatment strategies.

Citation Format: Hubert Tseng, Robert J. Amato, Reynolds Brobey, Cristian Ionescu-Zanetti, Jeff Jensen, Jacob Gage, Pujan Desai, Angela Liao, Mehdi Dehghani, Kevin Rosenblatt, Glauco Souza. Somatic mutation detection from liquid biopsy-derived cellular aggregates formed by magnetic 3D bioprinting. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B08.