bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3691: Met target inhibition-guided efficacy in preclinical models
Authors: Srivastava Apurva K. , Hollingshead Melinda G. , Govindharajulu Jeevan P. , Covey Joseph M. , Liston Dane , Peggins James , Bottaro Donald P. , Wright John J. , Kinders Robert J. , Tomaszewski Joseph E. , Doroshow James H. , Parchment Ralph E.
Published: May 2015

Background: A direct comparison of drug efficacy for the multiple agents currently in clinical development targeting MET-driven cancers would be useful for the selection of optimal treatment options. Previously, we utilized validated MET pharmacodynamic (PD) assays to compare the time course of phosphorylated-MET (pMET) suppression for five MET inhibitors (ASCO 2013). In the current study, we selected three candidates that demonstrated potent MET inhibition to compare anti-tumor efficacy.

Methods: PD time course and tumor PK data were utilized to simulate a dosing schedule anticipated to produce >90% pMET suppression in a SNU5 gastric cancer xenograft model. From these data, dosing schedules of 44 mg/kg (Q12H) cabozantinib, 12.5 mg/kg (Q12H) EMD1214063, and 16.5 mg/kg (QD) foretinib were chosen to achieve necessary tumor exposure to suppress pMET. Once tumors reached a 150±50 mm3 size, drugs were administered continuously for 21 days and tumor volumes were measured intermittently for 62 days. To measure pMET suppression, tumor quadrants were collected at 4, 12, and 24 hrs from all treatment groups after dose 1 (day 1) and on day 8 (after dose 8 of foretinib or dose 14 of cabozantinib and EMD1214063).

Results: Intact MET levels (pM/μg protein) were approximately 70% lower than vehicle controls at all day 8 collection points for all three drugs. The pY1234/35MET/MET ratios were 89%-99% (p<0.001) lower than vehicle controls on day 8 at 4 and 12 hrs post-dose for all three drugs. Compared to pretreatment levels, tumor volumes were reduced by 80-90% for all three drugs within 10-15 days post therapy and remained regressed until 13-20 days after treatment was stopped. Tumors then slowly regrew, but remained approximately 80% smaller than the vehicle group on day 62 (end of study).

Conclusions: These studies demonstrate that PD response-guided regimens delivered drug doses that were lower than efficacious doses described previously (except cabozantinib), but effectively reduced tumor volume in SNU5 xenografts. The three MET inhibitors showed comparable anti-tumor efficacy when accompanied by equivalent pMET inhibition. Our data also affirms the utility of the MET PD assays to guide dose ranging studies. Funded by NCI Contract No HHSN261200800001E.

Citation Format: Apurva K. Srivastava, Melinda G. Hollingshead, Jeevan P. Govindharajulu, Joseph M. Covey, Dane Liston, James Peggins, Donald P. Bottaro, John J. Wright, Robert J. Kinders, Joseph E. Tomaszewski, James H. Doroshow, Ralph E. Parchment. Met target inhibition-guided efficacy in preclinical models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3691. doi:10.1158/1538-7445.AM2014-3691
Abstract 3694: Relationship between in vivo antitumor activity of ADC and payload release in preclinical models
Authors: Li Fu , Zhang Xinqun , Emmerton Kim , Jonas Mechthild , Setter Jocelyn , Arthur Bill , Okeley Nicole , Lyon Robert , Benjamin Dennis , Law Che-Leung
Published: May 2015

Antibody-drug conjugates (ADC) deliver cytotoxic payloads to target cells via receptor-mediated internalization. We have demonstrated that SGN-35 (cAC10-vcMMAE) binds to CD30 on target cells and releases intracellular monomethyl auristatin E (MMAE) in vitro, contributing to potent killing of CD30+ cells. Using a set of auristatin-based ADCs, we evaluated whether the intratumoral concentration of the released payload is a predictive indicator of antitumor activity in preclinical models.

We first treated CD30+, anaplastic large cell lymphoma (ALCL) Karpas 299 tumors with SGN-35 or a non-binding control ADC (IgG-vcMMAE). The plasma ADC and MMAE PK measurements were similar in SGN-35 and IgG-vcMMAE treated tumor-bearing animals. In contrast, SGN-35 generated 5-fold greater intratumoral MMAE exposure than IgG-vcMMAE, which correlated with tumor regression observed in the SGN-35- treated group.

We then examined the relationship between released MMAE and in vitro cytotoxicity by targeting different receptors expressed on the same cells. The lymphoma cell line L82 expresses CD30, CD70, and CD71 receptors. Although auristatin-based ADCs targeting each of these targets has a different IC50, the intracellular MMAE was similar when 50% growth inhibition was achieved (approximately 200 nM). In vivo, CD30, CD70, or CD71-directed ADCs demonstrated similar antitumor activity for a given dose level (0.5, 1, or 3 mg/kg). Concordantly, equal intratumoral MMAE concentrations were observed for a given dose level, regardless of the antigen target.

The cell permeable MMAE has been shown to mediate bystander killing in vitro. To evaluate whether intratumoral MMAE mediates bystander killing in vivo, an admixed tumor model consisting CD30+ and CD30- Karpas 299 cells was treated with SGN-35. As predicted, both CD30+ and CD30- tumor cells were killed, presumably due to intratumoral diffusion of MMAE across cell membranes. In contrast, a conjugate with a highly polar form of auristatin (MMAF) was unable to kill the CD30-negative cell population of the admixed tumors.

These preclinical results provide evidence that the concentration of released MMAE within the tumor correlates with the ADC antitumor activity in vitro and in vivo. Moreover, the in vivo bystander activity of MMAE may improve utility of this chemotype in tumors with heterogeneous antigen expression.

Citation Format: Fu Li, Xinqun Zhang, Kim Emmerton, Mechthild Jonas, Jocelyn Setter, Bill Arthur, Nicole Okeley, Robert Lyon, Dennis Benjamin, Che-Leung Law. Relationship between in vivo antitumor activity of ADC and payload release in preclinical models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2014-3694
Abstract 3696: Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations
Authors: Hu Shuiying , Bruijn Peter D. , Mathijssen Ron H.j. , Baker Sharyn D. , Sparreboom Alex
Published: May 2015

Background: Several tyrosine kinase inhibitors (TKIs) evaluated in combination regimens with docetaxel, including axitinib, pazopanib, and sorafenib, can increase the systemic exposure to taxane in cancer patients by an unknown mechanism. We previously reported that differential expression of organic anion transporting polypeptides of the OATP1B-family in the human liver regulate the initial, rate-limiting step in the elimination of docetaxel, in advance of metabolism. In the current study, we tested the hypothesis that these interactions are mediated by the hepatic uptake transporter OATP1B1 using in vitro and in vivo models.

Materials and Methods: The influence of 16 approved TKIs on transport was studied in vitro using HEK293 cells expressing OATP1B1 or its mouse equivalent OATP1B2, determined by assessing the effect of TKIs on the intracellular accumulation of estradiol-17β-D-glucuronide, or docetaxel. Contribution of this process to an interaction with docetaxel in vivo was determined by pharmacokinetic studies using mice that are knock-out for OATP1B2 or knock-in for OATP1B1.

Results: Axitinib, nilotinib, pazopanib, and sorafenib were identified as potent inhibitors of OATP1B1 (>90% inhibition) when using estradiol-17β-D-glucuronide as a prototypical model substrate. As a representative of this class of TKIs, sorafenib was further evaluated and found to also potently inhibit OATP1B1-mediated transport of docetaxel with a half-inhibitory maximum concentration (IC50) of 6.96 nM, and almost completely inhibited the function of OATP1B1 and OATP1B2 at a concentration readily achievable in humans and mice. While OATP1B2-deficiency was associated with significantly increased exposure to docetaxel, as measured by peak plasma concentration (Cmax, P<0.05) and area under the curve (AUC, P<0.001), co-administration of a single or multiple oral dose of sorafenib did not result in statistically significantly altered exposure to docetaxel in either wildtype or OATP1B2 (-/-) mice.

Conclusion: These findings support a direct contribution of OATP1B1 in previously recorded pharmacokinetic interactions between TKIs and docetaxel, which can be predicted from a simple in vitro experiment. Our failure to reproduce this interaction in mice suggests that caution is warranted when attempting to extrapolate in vivo findings to a clinical scenario.

Citation Format: Shuiying Hu, Peter De Bruijn, Ron H.j. Mathijssen, Sharyn D. Baker, Alex Sparreboom. Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2014-3696
Abstract 3697: SIRT2 is a modulator of response to targeted therapies through regulation of MEK kinase activity
Authors: Bajpe Prashanth K. , Prahallad Anirudh , Horlings Hugo , Nagtegaal Iris , Beijersbergen Roderick , Bernards Rene
Published: May 2015

Resistance to targeted therapies is a major problem in cancer treatment. The Epidermal Growth Factor Receptor (EGFR) antibody drugs are effective in a subset of colorectal cancers, but the molecular mechanisms of resistance are understood poorly. Genes involved in epigenetic regulation are frequently deregulated in cancer, raising the possibility that such genes also contribute to drug resistance. To address this question, we compiled an shRNA library for chromatin modifying enzymes, chromatin remodelers, and many other factors associated with chromatin regulation. Using this library, we performed a loss of function genetic screen in colon cancer cell lines sensitive to EGFR inhibitors. We identified SIRT2, a NAD+ dependent deacetylase, as a modulator of the response to EGFR inhibitors in colon and lung cancer. Mechanistically, loss of SIRT2 expression enhanced MEK acetylation, resulting in increased ERK activity. Conversely, overexpression of SIRT2 led to reduced phospho ERK and inhibition of growth. SIRT2 loss also conferred resistance to BRAF and MEK inhibitors in BRAF mutant melanoma and KRAS mutant colon cancers, respectively. These results warrant a further investigation into a potential role of SIRT2 in resistance to drugs that act in the Receptor Tyrosine Kinase-RAS-RAF-MEK-ERK signaling pathway.

Citation Format: Prashanth K. Bajpe, Anirudh Prahallad, Hugo Horlings, Iris Nagtegaal, Roderick Beijersbergen, Rene Bernards. SIRT2 is a modulator of response to targeted therapies through regulation of MEK kinase activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3697. doi:10.1158/1538-7445.AM2014-3697
Abstract 37: Bone metastases in renal cell carcinoma are predicted by characteristics of the primary tumor
Authors: Schneider Elke , Haber Tobias , Roos Frederick C. , Hampel Christian , Junker Kerstin , Thüroff Joachim W. , Brenner Walburgis
Published: May 2015

Introduction: The poor prognosis of renal cell carcinoma (RCC) is caused by a high risk of metastasis. 30 % of all RCC patients develop bone metastases during course of disease. The outcome of RCC patients with bone metastases is poor so that a prediction would be of advantage. The aim of this study was to elucidate the mechanisms that lead to an organ specific metastasis of RCC in bones and probably predict bone metastasis.

Methods: In 30 RCC tissue specimens and 9 RCC primary cell lines collected from patients who had developed no metastases, lung or bone metastases within 5 years after nephrectomy (each 10 RCC tissue specimens, each 3 RCC cell lines) expression and/or activity of 46 cellular signalling molecules were quantified by a phospho kinase array and Western blot analyses. As steps of metastasis, migration of the primary RCC cells was analysed in a Boyden chamber with 10 µg/ml fibronectin as chemotaxin, adhesion to extracellular matrix compounds fibronectin, collagen I and IV was determined after cell staining with crystal violet.

Results: In RCC tissue specimens and primary cells from patients who developed bone metastases a higher expression of alpha5 integrins, higher activity of Akt and FAK as well as a lower expression of PTEN was detected, compared to those from patients without or with lung metastases. According to these results we observed an enhanced chemotactic migration (13-fold) of bone metastatic RCC cells and adhesion to fibronectin (6-fold) or collagen type I (8-fold) - both components of the bone matrix.

Conclusion: These results show that specific characteristics like the expression and activity of cell signalling mediators and the cellular behaviour of the primary tumor determine the location of the subsequent metastasis in RCC and could be used as prognostic marker for bone metastasis. This should be considered during follow-up care.

Funded by the Wilhelm Sander Foundation

Citation Format: Elke Schneider, Tobias Haber, Frederick C. Roos, Christian Hampel, Kerstin Junker, Joachim W. Thüroff, Walburgis Brenner. Bone metastases in renal cell carcinoma are predicted by characteristics of the primary tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 37. doi:10.1158/1538-7445.AM2014-37
Abstract 3705: Overcoming drug resistance in BRaf mutated melanoma cells
Authors: Samadani Ramin , Zhang Jun , Mackerell Alexander , Fletcher Steven , Shapiro Paul
Published: May 2015

Targeted inhibition of the ERK1/2 signaling pathway has received much attention after the transient success of the BRaf inhibitor, vemurafenib, in metastatic melanoma patients with the BRafV600E mutation. Approximately 50% of melanomas have an activating mutation to BRaf which drives cancer cell proliferation and survival through the ERK pathway. Therapies targeting BRaf or its downstream kinase MEK1/2 have demonstrated very promising initial clinical results. However, these treatments invariably lead to drug resistance through the activation of alternate pathways or mutations that circumvent the blockade and reactivate ERK1/2. Current efforts are centered on the use of combination therapies as well as the direct inhibition of ERK1/2 itself. The purpose of this study is to examine the effects of putative substrate-selective ERK inhibitors in various clinically relevant models of drug resistance. Specifically melanoma cell lines resistant to BRaf and MEK inhibitors, PLX-4032 and AZD-6244 respectively, were generated to test the efficacy of these novel compounds in cell viability assays. Furthermore, the potency of these molecules was evaluated in the context of activating mutations to MEK1 and drug resistant cell lines that overexpress MAP3K8 (the gene encoding COT/Tpl2). The data shows that these putative substrate-selective ERK inhibitors preferentially inhibit proliferation of cell lines with activated ERK1/2 signaling. In addition, these novel molecularly targeted therapies are equipotent in drug resistant versus non-resistant melanoma cells. Given these compounds' selectivity for BRaf mutated melanoma cells and their efficacy in drug resistance models, we submit these novel chemical entities as potential lead candidates for new salvage therapies in patients who have become resistant to currently available treatments. Moreover, these ATP-independent inhibitors target only select ERK1/2 functions and may be inherently less susceptible to developing their own drug resistance as compared to complete ablation of all ERK1/2 signaling as seen with ATP-competitive inhibitors.

Citation Format: Ramin Samadani, Jun Zhang, Alexander Mackerell, Steven Fletcher, Paul Shapiro. Overcoming drug resistance in BRaf mutated melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3705. doi:10.1158/1538-7445.AM2014-3705
Abstract 3711: Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors
Authors: Seipelt Irene , Schmidt Peter , Maerzhaeuser Helene , Gerlach Matthias , Jung Kai , Schuster Tilmann , Teifel Michael
Published: May 2015

The MAPK pathway represents a prime target for therapeutic intervention in cancer. Recent approvals of B-Raf and Mek inhibitors lead to exciting antitumor activities and survival benefits. However the observation of resistance to such Raf and Mek inhibitors has been documented in both clinical and preclinical trials. These phenomena stimulated further research into the regulation of the MAPK pathway and led to the suggestion to target the downstream kinase Erk, as well as to combine its targeting with Raf and Mek inhibitors.

Here we report that Erk inhibitors result in efficient growth inhibition of tumor cells that are resistant to Raf and Mek inhibitors. AEZS-134 - a highly potent and selective ATP competitive Erk inhibitor - overcomes the Raf inhibitor induced paradoxical cell activation and the aquired resistance to Mek inhibitors in tumor cells. We demonstrate potent antiproliferative activity of AEZS-134 in B-Raf wildtype, B-RafV600E mutant, Ras wildtype and K-Ras mutant tumor cell lines in comparison to common Raf inhibitors. Furthermore we show that AEZS-134 is efficacious in Mek inhibitor resistant Hct116 and MDA MB231 cells which have been well characterized in terms of Mek F129L allosteric binding pocket mutation, varying degrees of K-Ras amplification, cellular proliferation assays and MAPK pathway phosphorylation studies.

Our data provide a rational for new therapeutic opportunities by using Erk inhibitors in oncology. In order to maximize therapeutic benefit for patients and considering that Erk is directly downstream of B-Raf and Mek, targeting Erk may be more effective than targeting Raf or Mek in a variety of MAPK resistance settings. AEZS-134 and related compounds provide potent and selective tools to help address this question.

Development of the Erk inhibitors by Æterna Zentaris is integral part of our inhouse kinase research program comprising the investigation of different compounds for single Erk inhibition, single PI3K inhibition and dual kinase inhibitions of Erk and PI3K. All compounds are exclusively synthesized by our medicinal chemistry department.

Citation Format: Irene Seipelt, Peter Schmidt, Helene Maerzhaeuser, Matthias Gerlach, Kai Jung, Tilmann Schuster, Michael Teifel. Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2014-3711
Abstract 3719: Mechanisms of resistance to the second-generation alk inhibitor ap26113 in human npm-alk-positive anaplastic large cell lymphoma cells
Authors: Ceccon Monica , Mologni Luca , Giudici Giovanni , Piazza Rocco , Pirola Alessandra , Fontana Diletta , Gambacorti-Passerini Carlo
Published: May 2015

ALK is a tyrosine kinase receptor involved in a broad range of solid and haematological tumors. It has been shown that about 70-80% of ALK+ Anaplastic Large Cell Lymphoma (ALCL) cases, an aggressive Non Hodgkin T-cell lymphoma, are characterized by the traslocation t(2;5), originating the functional, aberrant oncogenic fusion protein NPM-ALK. Crizotinib was the first clinically relevant ALK inhibitor, now approved for the treatment of late stage and metastatic cases of lung cancer. Several patients soon developed Crizotinib resistance, mainly due to the appearance of new point mutations located in ALK kinase domain. Currently, other ALK inhibitors are available and already in clinical trial, hopefully representing the second line therapy able to overcome Crizotinib resistance. In this work we focused our attention on the more potent phase I/II dual ALK/EGFR inhibitor AP26113 (Ariad Pharm.), able to overcome the Crizotinib-resistant gatekeeper mutation L1196M. Two NPM-ALK+ human cell lines, KARPAS-299 and SUP-M2, were grown in four independent flasks in the presence of increasing doses of AP26113, so that eight cell lines able to grow in the presence of high AP26113 doses were selected. All cell lines show an AP26113 50% inhibitory concentration (IC50) value substantially higher than the one observed in parental cells, with a 130 to 1000-fold increase. All KARPAS-299 populations resistant to AP26113 show NPM-ALK overexpression, due to oncogene amplification, as the main cause of resistance, while SUP-M2 cells proliferating in the presence of the drug harbour several point mutations spanning the entire ALK kinase domain. In particular, we identified the following aminoacid substitutions: L1122V, , L1196M, S1206C and a double F1174V+L1198F mutation. The knowledge of the possible appearance of new clinically relevant mechanisms of drug resistance is a useful tool for the management of new TKI resistant cases.

Citation Format: Monica Ceccon, Luca Mologni, Giovanni Giudici, Rocco Piazza, Alessandra Pirola, Diletta Fontana, Carlo Gambacorti-Passerini. Mechanisms of resistance to the second-generation alk inhibitor ap26113 in human npm-alk-positive anaplastic large cell lymphoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3719. doi:10.1158/1538-7445.AM2014-3719
Abstract 3722: The HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitor crizotinib in both crizotinib-sensitive and crizotinib-resistant MET-driven renal tumor models
Authors: Miyajima Naoto , Akino Tomoshige , Tsuchiya Kunihiko , Maruyama Satoru , Abe Takashige , Shinohara Nobuo , Nonomura Katsuya , Neckers Len
Published: May 2015

Introduction: The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers including papillary renal carcinoma, making it a prime anti-cancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. Small molecule HSP90 inhibitors cause HSP90 to dissociate from its client proteins, resulting in their destabilization and eventual degradation. HSP90 inhibitors have been validated in numerous preclinical tumor models and have shown promising activity in several clinical trials. Given the distinct mechanisms of action of TKI and HSP90 inhibitors, we evaluated the synergistic effects of these two drugs on both TKI-sensitive and TKI-resistant MET-driven tumor models.

Methods: We tested efficacy of the MET TKI crizotinib, the HSP90 inhibitor ganetespib and their combination in MKN45, wild-type MET-overexpressed cell line and MET-Y1248H, TKI-resistant mutant MET- verexpressed cell line in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in xenograft models.

Results: MET is an HSP90-dependent kinase, and we show that HSP90 preferentially interacts with and stabilizes activated MET, whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET TKI show a preference for the inactive form of the kinase and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in TKI-sensitive MET-driven tumor models. Unexpectedly, low-dose ganetespib also partially restores crizotinib sensitivity, in vitro and in vivo, to cells and tumors expressing TKI-resistant MET mutants.

Conclusion: Our findings support the use of HSP90 inhibitors to overcome or delay the initiation of resistance to MET TKI, and they provide the basis for clinical evaluation of this combination in patients with MET-driven papillary renal cancer.

Citation Format: Naoto Miyajima, Tomoshige Akino, Kunihiko Tsuchiya, Satoru Maruyama, Takashige Abe, Nobuo Shinohara, Katsuya Nonomura, Len Neckers. The HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitor crizotinib in both crizotinib-sensitive and crizotinib-resistant MET-driven renal tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3722. doi:10.1158/1538-7445.AM2014-3722
Abstract 3725: HGF/c-Met axis drives cancer aggressiveness in the neo-adjuvant setting of ovarian cancer
Authors: Mariani Marisa , McHugh Mark , Petrillo Marco , Sieber Steven , He Shiquan , Andreoli Mirko , Fiedler Paul , Scambia Giovanni , Shahabi Shohreh , Ferlini Cristiano
Published: May 2015

Background: Ovarian cancer is the most lethal gynecologic malignancy. Recently, NACT has been clinically tested as alternative approach for the management of ovarian cancer patients. A biological predictor helpful in selecting patients for NACT would be desirable. Indeed, chemotherapy has a strong impact in modifying the biology of tumor and rendering it more aggressive. This study was aimed at identifying actionable mechanisms of resistance to NACT.

Methods: Expression of a panel of microRNAs was screened in a discovery set of 85 patients. Analysis of the potential targets was conducted in the same RNAs by calculating significant correlations between microRNAs and genes. Quantitative fluorescent immunohistochemistry was employed in a validation set of 109 patients. Pre and post-NACT specimens were analyzed in multiple cores of tumor from formalin fixed paraffin embedded material (FFPE).

Results: MiR-193a-5p was significantly overexpressed in the NACT setting. Analysis of its potential targets demonstrated that this microRNA is also significantly correlated with the expression levels of HGF and MET genes. Analysis of protein expression in samples taken before and after NACT demonstrated that both HGF and c-Met are increased after NACT. Patients who relapse shortly after NACT exhibited the highest relative basal expression of both HGF and c-Met, while the opposite phenomenon was observed in the best responders.

Conclusion: These findings demonstrate that NACT refractory patients express activation of the HGF/c-Met pathway. HGF/c-Met expression may help select eligible patients for this modality of treatment. Moreover, inhibitors of this pathway may improve the efficacy of NACT.

Citation Format: Marisa Mariani, Mark McHugh, Marco Petrillo, Steven Sieber, Shiquan He, Mirko Andreoli, Paul Fiedler, Giovanni Scambia, Shohreh Shahabi, Cristiano Ferlini. HGF/c-Met axis drives cancer aggressiveness in the neo-adjuvant setting of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3725. doi:10.1158/1538-7445.AM2014-3725
Abstract 3727: Drug screen on six co-cultured GBM cell lines using a multiplex luciferase assay
Authors: Rijn Sjoerd van , Hiddingh Lotte , Wurdinger Thomas , Narayan Ravi
Published: May 2015

Despite the addition of temozolomide (TMZ) to the glioblastoma (GBM) treatment regimen, disease outcome is still poor and additional therapeutic options are necessary. One major obstacle in the GBM treatment is TMZ resistance of the tumor cells resulting in relapse of the disease with no alternative treatments known. In an effort to discover additional treatment modalities we are performing drug screens on GBM cell lines, using wild-type variants and acquired TMZ resistant variants of these cell lines.

To be able to screen a range of different cells, we adapted a multiplex bioluminescence reporter assay developed in our lab. This bioluminescent reporter assay is based on the Gaussia luciferase (Gluc) fused to an epitope tag. Different cell lines can be equipped with Gluc fused to different epitope tags (Gluc-tag) so that each cell lines expresses Gluc and a cell line specific epitope tag. Antibody immunoprecipitation allows us to separate the different Gluc-tags and quantify the relative activities of the different cell lines. Activity of each specific Gluc-tag represents the relative number of a specific cell line in the mixed population. Since Gaussia luciferase and the epitope tag is secreted by the cells, we can detect the cell line specific reporters ex vivo in the culture medium or animal blood.

Using this novel method in a drug screen we aim to identify additional subtype specific therapy strategies for GBM braintumors.

Citation Format: Sjoerd van Rijn, Lotte Hiddingh, Thomas Wurdinger, Ravi Narayan. Drug screen on six co-cultured GBM cell lines using a multiplex luciferase assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2014-3727
Abstract 3731: Luminescent cell health assays for tumor spheroid evaluation
Authors: Valley Michael P. , Kupcho Kevin R. , Zimprich Chad A. , Niles Andrew L. , Cali James J. , Kelm Jens M. , Moritz Wolfgang , Lazar Dan F.
Published: May 2015

Microtissues produced in 3D cell culture are much more representative of actual living tissue compared to monolayers produced in 2D cell culture. In fact, in the area of oncology research, multicellular tumor spheroids are considered an excellent platform for testing drug delivery and efficacy. As the necessity for established 3D cell culture models rises, there is also a need for convenient assays that have been specifically demonstrated to be effective for use with 3D microtissues. The more complex architecture of 3D microtissues demands increased lytic effectiveness and reagent penetration, characteristics that are often only minor considerations for reagents designed for 2D cell culture. Here we report on a variety of bioluminescent and fluorescent cell-based assays applied to hanging-drop spheroids produced from HCT116 colon cancer cells. The first assay to be described is an ATP detection reagent for measuring cell viability. This reagent has both an improved formulation and an optimized assay protocol and has clear advantages over other viability assays. Other cell health assays will also be described, including reagents that measure cell death, apoptosis, mechanistic cytotoxicity, or reporter gene expression. These additional assays do not require a change in formulation, but do require new protocols in order to optimize their effectiveness when applied to 3D microtissues. As with their application to cells in 2D culture, these “add-mix-measure” reagents are robust and amenable to both low- and high-throughput applications.

Citation Format: Michael P. Valley, Kevin R. Kupcho, Chad A. Zimprich, Andrew L. Niles, James J. Cali, Jens M. Kelm, Wolfgang Moritz, Dan F. Lazar. Luminescent cell health assays for tumor spheroid evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2014-3731
Abstract 374: A novel sequencing method for genome-wide profiling of 5-hydroxymethylcytosine with single-base resolution
Authors: Sun Xueguang , Chung Tzu-Hung , Chew Yap Ching , Tan Darany , Jia Xi-Yu
Published: May 2015

5-hydroxymethylcytosine (5hmC) is an epigenetic mark abundant in embryo stem cells and brain tissues. The exact biological functions of 5hmC are still under close investigation although several lines of evidence have indicated it could be involved in active DNA demethylation. Meanwhile, extensive studies have been carried out to determine its genomic distribution. A number of approaches have been developed using either affinity based enrichment, such as hMeDIP, that rely on antibody and other specific binding proteins to target 5hmC, or modified bisulfite sequencing, namely oxidative bisulfite sequencing (OxBS) and TET assisted bisulfite sequencing (TAB-sequencing). However, all those methods have limitations which hamper their application. For example, affinity based methods lack single base resolution while modified bisulfite sequencing methods require efficient chemical or enzymatic oxidation which cannot be easily achieved or guaranteed. As an alternative, we have developed a novel genome-wide sequencing method that utilizes an enzyme based modification approach coupled with bisulfite-sequencing for detecting 5hmC. This methodology allows quantification of 5hmC levels with single CpG resolution and can also be employed for locus-specific assays. Using this method, we were able to map and quantify 5hmC sites at the genomic scale for several different biological samples. This novel method can determine the exact location and abundance of 5hmC, which will facilitate our understanding of 5hmC in regulating gene expression in different biological contexts.

Citation Format: Xueguang Sun, Tzu-Hung Chung, Yap Ching Chew, Darany Tan, Xi-Yu Jia. A novel sequencing method for genome-wide profiling of 5-hydroxymethylcytosine with single-base resolution. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2014-374
Abstract 3744: High-throughput in vitro screening of glioma stem cell lines: Evaluation of over 350 tyrosine kinase inhibitors
Authors: Thomas Craig , Liang Ji , Piao Yuji , Nguyen Nghi , Sulman Erik , Stephan Clifford , Yung Alfred , Groot John F. de
Published: May 2015

Despite the availability of hundreds of drugs, there is little data on the efficacy of these agents in the extremely heterogeneous populations of tumor cells observed in glioblastoma. A high-throughput compound-screening assay was used to identify drug sensitivities in a panel of glioma stem cell (GSC) lines to 350 tyrosine kinase inhibitors (GSK PKIS1) with varying cellular targets and mechanisms of kinase inhibition. Twelve glioma stem cell lines, which are representative of the classic TCGA molecular subtypes, were screened. Cell lines were screened in 384-well plates at a density of 1000 cells/well with drug concentrations of either 5 or 0.5 µM. Cell viability measurements were taken using Cell-Titer Glo five days after drug treatment and compared to control wells to calculate percentage inhibition. Compounds were defined as highly active (>90 % inhibition at 5µM; > 50% inhibition at 0.5µM), moderately active (>70% inhibition at 5µM; >30% inhibition at 0.5µM), or weakly active (>50% inhibition at 5µM; >15% inhibition at 0.5µM.) We identified three compounds that were highly active against eleven cell lines, three more compounds that were highly effective against at least seven cell lines, and seven additional compounds that were highly effective against at least four cell lines. The compounds in the screen were further analyzed by testing their inhibition efficacy against 220 kinases. A separate set of compounds containing molecularly targeted agents to TGF-beta, STAT3, PI3K and Notch were used for comparison of drug efficacy. Ongoing experiments are being performed to fully characterize the molecular phenotypes of the different GSC's by analyzing RNAseq, miRNA, the whole exome, the proteome, DNA methylation, and copy number variation. Integrating this information with the kinase and growth inhibition data will allow us to have a complete picture of the underlying mechanisms which drive resistance/sensitivity in GSC's. After determining which kinases are the most effective targets for the treatment of glioblastoma, promising compounds will be validated in intracranial in vivo xenograft models.

Citation Format: Craig Thomas, Ji Liang, Yuji Piao, Nghi Nguyen, Erik Sulman, Clifford Stephan, Alfred Yung, John F. de Groot. High-throughput in vitro screening of glioma stem cell lines: Evaluation of over 350 tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3744. doi:10.1158/1538-7445.AM2014-3744
Abstract 3745: Optimization of an assay for the detection of phosphorylated FAK by immunohistochemistry in formalin-fixed, paraffin-embedded human tissue and cell lines
Authors: Dauffenbach Lisa M. , Sia Gela C. , Cash Patricia A. , Girees Sherif K. , Lim Ryan S. , Zheng Jianping , Olsen Eric P. , Richeh Rana , Kerfoot Christopher A.
Published: May 2015

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase with elevated expression in most human cancers, most notably in invasive metastasis. FAK is involved in cell adhesion, motility, and apoptosis, and it is a target for oncology therapeutics. FAK activation occurs by phosphorylation at Tyr397 in response to integrin clustering caused by cell adhesion. The development of a sensitive and specific immunohistochemical assay for phosphorylated FAK (pFAK) is important for pharmacodynamic biomarker studies. The purpose of this study was to investigate 4 commercially available anti-pFAK antibodies in human tissues and cell lines. Antibodies included 3 rabbit monoclonals (EP2160Y, 141-9, and 31H5L17) and 1 rabbit polyclonal. Antibody specificity was evaluated in a variety of cell lines (BxPC3, HCT-116, LOVO, MDA-MB-231, and MiaPaca2) treated with 2 FAK inhibitors: TAE226 (NVP-TAE226) and PF-562271. Decreased immunohistochemical staining for pFAK was observed in post-treatment samples of cell lines with baseline pFAK expression. TAE226 was more potent at inhibiting pFAK staining. Membrane staining was observed with the rabbit polyclonal and rabbit clone EP2160Y only. Each of the 4 antibodies was tested in human breast cancer tissues using tissue microarrays created by Mosaic Laboratories. The percentage of breast cancer samples that were pFAK positive was 96% with clone EP2160Y, 83% with clone 141-9, 92% with clone 31H5L17 and 96% with the polyclonal. The percentage of cells that were positive within each breast cancer sample ranged from 63% to 83%, depending on the antibody used (standard deviations between 30% and 44%). Immunohistochemistry with the polyclonal and clone EP2160Y antibodies demonstrated membrane and cytoplasmic staining, while clones 141-9 and 31H5L17 demonstrated primarily cytoplasmic staining. Based on reduction of pFAK staining in treated cells and acceptable performance in human cancer tissues, pFAK immunohistochemistry is “fit for purpose” for evaluation of FAK inhibitor activity in pre- and post-treatment clinical trial tissue biopsies.

Citation Format: Lisa M. Dauffenbach, Gela C. Sia, Patricia A. Cash, Sherif K. Girees, Ryan S. Lim, Jianping Zheng, Eric P. Olsen, Rana Richeh, Christopher A. Kerfoot. Optimization of an assay for the detection of phosphorylated FAK by immunohistochemistry in formalin-fixed, paraffin-embedded human tissue and cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2014-3745
Abstract 3751: The potential role of CXCR4 in cisplatin associated resistance in esophageal carcinoma
Authors: Zhao Yue , Wang Yan , Bruns Christiane
Published: May 2015

Introduction

CXC chemokine receptor 4 was found dys-regulated in many different types of human cancers and correlated with poor prognosis of tumor metastasis and resistance to chemotherapy. Limited data show the effect of CXCR4 on the esophageal carcinoma cells, especially with influence of chemotheraputuc reagent-cisplatin.

Methods

CXCR4 was detected by FACs analysis in esophageal cell lines OE19, OE21, OE33, PT1590 and LN1590. Corronsponding cisplatin resistant cells were established after selection of cisplatin treatment and further confirmed by IC50 and ERCC1. SDF-1α and AMD3100 were used as CXCR4 stimulattion or inhibition. Subcutanous model of OE19 injection in Balb/c nude mice was established and followed by treatment with cisplatin twice a week via i.p injection. Tumors were collected for the further evaluation.

Results

Esophageal cell line OE19, PT1590 and LN1590 have detetable amount of CXCR4+ subpopulation cells as high as 2.8%±0.9, 0.7%± 0.9 and 1.0%±0.6, respectively. OE19, PT1590 and LN1590 cisplatin resistant subline displayed increased IC50 value of 48h cisplatin treatment from 6.0±1.3 to 12.2±1.0, 1.4±0.7 to 9.1±0.7 and 1.4±0.6 to 7.2±0.6 ug/ml along with a significant overexpression of ERCC1. Interestingly only OE19 and LN1590 cisplatin resistant cell lines showed a significant enreichment of CXCR4+ cells ( 12.1%± 2.5 Vs 2.8%±0.9; 4.2%± 1.1 Vs1.0%±0.6, p<0.05) as compared to parental cells. Meanwhile, side population cells were also increased in those cisplatin resistant variants. There is no difference on tumor weight in control group and cisplatin treatment group in vivo. The relative expression of CXCR4 will be further evaluated by realtime PCR on mRNA level and IHC on protein level.

Conclusion

Overexpression of CXCR4 is significantly associated with cisplatin-based chemotherapy resistance and might be a prognostic factor in esophageal cancer. Inhibition of CXCR4 might be a strategy to address the chemoresistance of EAC cell lines.

Citation Format: Yue Zhao, Yan Wang, Christiane Bruns. The potential role of CXCR4 in cisplatin associated resistance in esophageal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3751. doi:10.1158/1538-7445.AM2014-3751
Abstract 3755: Restoring sensitivity to cisplatin or TRAIL by targeting drug resistance master regulators in endometrial cancer
Authors: Meng Xiangbing , Yang Shujie , Wang Xinjun , Jia Yichen , Zhang Yuping , Leslie Kimberly K.
Published: May 2015

Understanding the molecular mechanisms of drug resistance is vital to enhance the efficacy of current therapeutic regimens. In particular, metadherin (MTDH) and FOXM1 overexpression has been independently demonstrated to have a critical role in chemoresistance. Overexpression of MTDH correlates with poor clinical outcome in many cancers, including endometrial cancer. Our previous study showed that endometrial cancer cells are sensitive to the combination treatment of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) with histone deacetylase (HDAC) inhibitor LBH589, an effect that could be enhanced by depletion of MTDH. In this study, we focused on the therapeutic strategies to restore sensitivity to chemotherapeutic agent cisplatin or apoptosis-inducing TRAIL by disrupting drug resistance genes FOXM1 or MTDH. CUDC907 is a novel investigational dual inhibitor designed to target PI3K and HDACs. The high frequency of PI3K /PTEN/AKT pathway dysregulation in endometrial cancer suggests that CUDC907 will also be effective in this disease. Indeed, CUDC907 potently induced apoptosis when combined with TRAIL in MTDH-depleted endometrial cancer cells. Cisplatin is a frontline treatment for advanced endometrial cancers. However, drug resistance causes treatment failure. Although the detailed mechanisms of drug resistance are still elusive, gradual induction of FOXM1 and its downstream target gene network may be involved in the development of resistance to cisplatin. We found that co-treatment with cisplatin and the Nedd8 conjugation system inhibitor MLN4924 restored sensitivity to cisplatin by down-regulating drug resistant gene FOXM1. These findings imply that simultaneously depleting drug resistant master genes, such as MTDH and FOXM1, is a potentially novel avenue for reversing therapeutic resistance to current chemotherapy and targeted therapies in patients with endometrial cancer.

Citation Format: Xiangbing Meng, Shujie Yang, Xinjun Wang, Yichen Jia, Yuping Zhang, Kimberly K. Leslie. Restoring sensitivity to cisplatin or TRAIL by targeting drug resistance master regulators in endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2014-3755
Abstract 3757: Vacuolar ATPase ‘a’ subunit mediates platinum resistance in ovarian cancer
Authors: Kulshrestha Arpita , Katara Gajendra K. , Pamarthy Sahithi , Gilman-Sachs Alice , Beaman Kenneth D.
Published: May 2015

Development of resistance to platinum compounds significantly hinders successful ovarian cancer treatment. Understanding the molecular alterations in chemo-resistance is important for developing therapeutic strategies for reversal of resistance. Vacuolar-H + −ATPases (V-ATPases) are multi-subunit proton pumps that play key roles in regulating cytosolic/extracellular pH and drug tolerance in tumor cells. The present study begins to decipher the role of V-ATPase `a’ subunit isoforms (major pH sensing unit) in cisplatin resistance. For this, cisplatin resistant human ovarian cancer cell lines A2780 and TOV112D were generated in lab by stepwise increase in drug pressure. Comparative expression profiling revealed that V-ATPase `a` subunit levels correlated with cisplatin resistance and each of the a1,a2 and a3 isoforms were significantly up regulated in the resistant lines. Isoform-specific shRNAs were employed to selectively and stably reduce mRNA levels for VATPase ‘a’ subunit in cisplatin resistant cell lines and determine their effect on cisplatin/carboplatin sensitivity, DNA damage and apoptosis. V-ATPase a2 or a3 isoform knocked down resistant lines exhibited sensitization to cisplatin and carboplatin. In vitro effects showed increased DNA damage by forming platinum DNA adducts which led to higher levels of apoptosis. In conclusion, inhibition of V-ATPase `a` subunit expression by monoclonal antibodies or V-ATPase inhibitors could serve as a therapeutic strategy to overcome cisplatin resistance and has a potential as a combination therapy along with platinum drugs for treatment of chemo-resistant ovarian carcinoma.

Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth D. Beaman. Vacuolar ATPase ‘a’ subunit mediates platinum resistance in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3757. doi:10.1158/1538-7445.AM2014-3757