Non-responder Cell Research Articles

Abstract 4531: Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study

Abstract ZL-1211, a humanized monoclonal IgG1 antibody targeting CLDN18.2, induces cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). We developed ex vivo ADCC and CDC assays to study the mechanism of actions of ZL-1211. Our preclinical data has demonstrated greater antitumor activities than the clinical leading benchmark via enhanced ADCC by mutation in Fcγ domain and potent CDC activities. We used those ex vivo assays to further profile the clinical specimens from patients collected at baseline (pre-infusion) and the following cycles (post-infusion) in our phase 1 study (NCT05065710). Preliminary results in clinical samples indicated that baseline ADCC and CDC capabilities were correlated with anti-tumor activity of ZL-1211 in patients with CLDN18.2-positive locally advanced or metastatic solid tumors, suggesting probable predictive biomarkers. Results also showed that changes in circulating ZL-1211 mediated ADCC and CDC activities were dose-dependent and in accordance with the pharmacokinetic (PK) profile. In addition, we performed differential gene expression analysis and real-time PCR to compare gene expression in ZL-1211 responder and non-responder PDX models and CCLE cell lines. Our findings reveal that several immune-related genes may serve to predict the responses to ZL-1211 treatment. Other potential predictive biomarkers such as Fcγ receptor CD16A polymorphism and baseline stromal CD8 expression were also evaluated in clinical samples. Taken together, analytical approaches developed and validated from preclinical studies were successfully translated to characterize clinical samples in the current Phase 1 trial. The exploratory analysis indicated the clinical relevance of multiple biomarkers and their potential as predictive biomarkers of ZL-1211. Citation Format: Zhen Luo, Xiao Wang, Xuehuo Zeng, Jiaqing Yi, Tegan Burns, Herman Liu, Xinyu Zhang, Wenyu Liu, Karl Hsu, Hua Gong. Clinical translation of biomarkers for ZL-1211, an anti-CLDN18.2 antibody, in patients with advanced solid tumors from a phase I dose-escalation study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4531.

Functional Profiling of Head and Neck/Esophageal Squamous Cell Carcinoma to Predict Cetuximab Response.

Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting antibody, remains the only Food and Drug Administration-approved targeted therapy for squamous cell carcinoma (SCC) of head and neck/esophagus. However, in clinical trials, cetuximab only benefited a subset of patients and frequently caused toxicity. Predicting which patients respond to cetuximab remains unsolved. The authors sought to identify predictive biomarkers in EGFR signaling and autophagy pathways, which may be impacted by cetuximab under certain treatment conditions. Methods: In vitro responses of SCC cell lines to cetuximab under various nutrient conditions were assessed by WST-8 growth assay. Functional profiles of several EGFR signaling biomarkers were investigated by Luminex-based assays and corroborated with immunoblots. Autophagy markers were analyzed with immunoblots. Results: In vitro growth response assays identified cetuximab responder and nonresponder cell lines. Optimal growth conditions and growth factors enhanced responses, and even reversed nonresponsiveness in some cell lines. Strong correlation was found between response in growth assays (reference assay) and dynamic changes in p-Erk1/2 and LC3-II (index assays). Conclusions: This study indicates that nutrient modification may enhance cetuximab response in SCC patients. Biomarker results strengthen the hypothesis that dynamic biomarkers can be used to predict patient response to cetuximab. Future studies are warranted to test in more complex samples including patient-derived tumor tissues.

OTME-14. TGF-beta signaling in microtube formation of glioblastoma

Microtubes (MTs) are cytoplasmic extensions of glioma cells serving as important cell communication structures while also promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas, while they are uncommon in chemosensitive IDH mutated and 1p/19q co-deleted oligodendrogliomas.By performing a bioinformatics analysis on data from The Cancer Genome Atlas (TCGA) we identified the TGF-b pathway as being distinctly upregulated in glioblastomas compared to oligodendrogliomas, making this a signaling pathway potentially involved in MT formation. Based on patient-derived GBM stem cell line models we demonstrated that stimulation of TGF-b increased MT formation, while inhibition of TGF-b reduced MT formation. MT formation was verified by expression of GAP43 and nestin, which have previously been shown to be important structural proteins of MTs.Interestingly, we also observed a responder/non-responder relationship between GBM cell lines P3 and GG16/ GG6 regarding MT formation upon TGF-b stimulation. To determine downstream signaling mediators of the TGF-b pathway crucial for MT formation, we subsequently performed RNA sequencing of these cell lines. From the 34 initial candidates common to responders, but absent in non-responders, only 3 genes were left after filtering through TCGA data and in vivo RNA sequencing data of a GBM xenograft model derived from P3.Thrombospondin 1 (TSP1) emerged as the most interesting candidate as we have previously shown that transcription of this gene is activated by TGF-b/SMAD signaling and TSP1 also promotes invasiveness of GBM. TSP1 was upregulated by TGFB1 stimulation in responder cells and promoted MT formation. Transcriptional activation of TSP1 was absent in the non-responder cell line GG6 and could be reversed in the responder cell line P3 by TSP1 shRNAs in vitro and in vivo. Thus, TSP1 was experimentally verified as an important mediator of microtube formation downstream of TGF-b signaling.

Abstract 5730: A new polyphenol prebiotic isolated from Myrciaria dubia improves gut microbiota composition and increases anti-PD-1 efficacy in murine cancer models

Abstract Introduction The gut microbiota influences immune checkpoint inhibitors (ICI) efficacy, and immunogenic commensals bacteria such as Akkermansia muciniphila (AM) and Ruminoccocus are enriched in ICI responder patients. Therefore, strategies to beneficially shift microbiota composition represent a novel therapeutic avenue. Myrciaria dubia (MD) is a polyphenol-rich berry prebiotic that has been recently shown to improve the gut microbiota composition and dampen inflammation in metabolic diseases. Methods Daily oral administration of MD or water was performed in MCA-205 (PD-1 sensitive) and E0071 (PD-1 resistant) murine tumor models treated with iso-control or anti-PD-1. 16s rRNA gene sequencing and qPCR were used to profile the murine fecal microbiota. Tumor immune infiltration was analyzed by flow cytometry and RNA sequencing. Bile acid levels were measured using liquid chromatography coupled to tandem mass spectrometry in mouse feces and plasma at sacrifice. MD supplementation was also tested in antibiotic-treated mice that had undergone a fecal microbiota transplantation (FMT) using feces from non-responder (NR) and responder (R) non-small cell lung cancer (NSCLC) patients. Finally, to determine the bioactive compound, reverse-phase high performance liquid chromatography was used to isolate fractions that were tested in the MCA-205 tumor model. Results In MCA-205, MD gavage alone significantly reduced tumor size and in both tumor models MD increased the efficacy of anti-PD-1 compared to water + anti-PD-1. The activity of MD was inhibited upon antibiotics administration. FMT using mouse feces previously treated with MD recreated the effect in MD-naïve mice. Increases in alpha diversity, and AM and Ruminococcaceae were observed in the feces of MD supplemented mice. Serum and fecal CA, αMCA primary and HDCA and DCA secondary bile acids were upregulated in mice treated with MD. The combination of MD + anti-PD-1 increased the frequency of CD8+/CD4+ Treg and ICOS+ expression on CD8+ T cells, while the depletion of the CD8+ cells inhibited the anti-cancer activity. Next, we validated that FMT from NSCLC ICI patients recreated the patients' phenotypes in the MCA-205 tumor model (R vs NR). Treatment with MD resorted anti-PD-1 anti-tumor efficacy post FMT from NR patients. Lastly, we isolated the bioactive polyphenol molecule from MD and confirmed its anti-cancer effect in vivo. Supplementation of AM and ruminococcus further increased anti-PD1 activity. Conclusion We isolated the bioactive polyphenol molecule from the MD berry and demonstrated that its potent adjuvant effect on anti-PD-1 was associated with a beneficial shift in microbiome and bile acid composition in a CD8+ T cell dependent manner. The modification of the gut microbiota with polyphenol supplementation provides a new therapeutic approach in immuno-oncology. Citation Format: Meriem Messaoudene, Florent Cauchois, Reilly Piedgeon, Khoudia Diop, Corentin Richard, Thibault Varin, Jocelyn Trottier, Olivier Barbier, Geneviève Pilon, Simon Turcotte, André Marette, Bastien Castagner, Bertrand Routy. A new polyphenol prebiotic isolated from Myrciaria dubia improves gut microbiota composition and increases anti-PD-1 efficacy in murine cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5730.

Sensitization of glioblastoma cells to TRAIL-induced apoptosis by IAP- and Bcl-2 antagonism

Due to the lack of effective treatments for glioblastoma (GBM), we here studied the responsiveness of GBM cell lines to the combination of death ligand, TRAIL and the IAP antagonist, TL32711 (Birinapant). Responses were highly heterogeneous, with synergistic apoptosis as well as treatment resistance observed. Caspase-8 and Bid, together with caspase-3, form a nonlinear signalling hub that efficiently induced apoptosis in responder cell lines. Cells resistant to TRAIL/TL32711 expressed low amounts of procaspase-8 and Bid and poorly activated caspase-3. We therefore hypothesised that improving caspase-8 activation or sensitising mitochondria to truncated Bid (tBid) could convert non-responder GBM cell lines to responders. Mathematical simulations of both strategies predicted mitochondrial sensitization to tBid would outperform enhancing caspase-8 activation. Indeed, antagonising Bcl-2 by ABT-199 allowed TRAIL/TL32711 response synergies to manifest in otherwise TRAIL resistant cell lines. These findings were further corroborated in experiments with a translationally relevant hexavalent TRAIL variant. Our study therefore demonstrates that a high caspase-8/Bid signature is associated with synergistic TRAIL/TL32711-induced apoptosis in GBM cells and outlines Bcl-2 antagonism as a highly potent intervention to sensitize highly TRAIL-resistant GBM cells to TRAIL/TL32711 combination treatment.

Whole exome sequencing identifies mTOR and KEAP1 as potential targets for radiosensitization of HNSCC cells refractory to EGFR and β1 integrin inhibition.

Intrinsic and acquired resistances are major obstacles in cancer therapy. Genetic characterization is commonly used to identify predictive or prognostic biomarker signatures and potential cancer targets in samples from therapy-naïve patients. By far less common are such investigations to identify specific, predictive and/or prognostic gene signatures in patients or cancer cells refractory to a specific molecular-targeted intervention. This, however, might have a great value to foster the development of tailored, personalized cancer therapy. Based on our identification of a differential radiosensitization by single and combined β1 integrin (AIIB2) and EGFR (Cetuximab) targeting in more physiological, three-dimensional head and neck squamous cell carcinoma (HNSCC) cell cultures, we performed comparative whole exome sequencing, phosphoproteome analyses and RNAi knockdown screens in responder and non-responder cell lines. We found a higher rate of gene mutations with putative protein-changing characteristics in non-responders and different mutational profiles of responders and non-responders. These profiles allow stratification of HNSCC patients and identification of potential targets to address treatment resistance. Consecutively, pharmacological inhibition of mTOR and KEAP1 effectively diminished non-responder insusceptibility to β1 integrin and EGFR targeting for radiosensitization. Our data pinpoint the added value of genetic biomarker identification after selection for cancer subgroup responsiveness to targeted therapies.

Abstract 1654: Development of subtype specific mouse models of bladder cancer

Abstract Introduction: High-grade, muscle-invasive bladder cancer has recently been shown to harbor intrinsic molecular subtypes with distinct biologic features. Current murine models of bladder cancer, including the prominent carcinogen induced model MB49, do not account for subtype specific characteristics, leaving a gap in available tools for understanding subtype specific differences in bladder cancer. We have developed and validated immunocompetent, subtype specific models of bladder cancer, and we have used these models to assess differential responses to immune checkpoint inhibition. Methods: Two distinct models of murine bladder cancer were developed in a C57BL/6 background. The UPPL models were generated through Pten/Trp53 conditional knockout in Uroplakin3a expressing cells. BBN models were generated through exposure of wild-type C57BL/6 mice to the carcinogen N-Butyl-N-(4-hydmoxybutyl)nitrosamine and subsequent generation of cell lines from spontaneous tumors. RNAseq was performed on several BBN and UPPL tumors and cell lines, with findings validated with flow cytometry and T/B cell receptor (TCR/BCR) amplicon sequencing of tumor infiltrating lymphocytes (TILs). Results: BBN and UPPL models reflected characteristics of human basal and luminal bladder cancers, respectively. BBN (basal) models demonstrated higher immune gene signature expression, with concordantly higher numbers of TILs compared to the UPPL (luminal) model (p < 0.0001). Two BBN and two UPPL models were assessed for response to anti-PD-1 therapy in vivo as syngeneic tumors grown in wild type C57BL/6 mice. One of the BBN lines (BBN963) demonstrated robust control of tumor growth in some animals, including multiple complete responses (p = 0.0003), but also tumors that progressed, leading us to characterize BBN963 as a mixed response model. The marked response to PD-1 blockade in BBN963 was associated with significantly higher sharing of TCR CDR3 sequences among TILs compared to sequences of the other tumors (p = 0.003). In addition, analysis of BBN963 tumors by flow cytometry demonstrated naïve and memory T cell phenotypes correlated with increased and decreased tumor sizes, respectively. Closer examination of individual BBN963 tumor responses to PD-1 blockade revealed distinct responder and non-responder infiltrating immune cell phenotypes. Responders demonstrated a less diverse B cell repertoire (p = 0.0043) with increased BCR CDR3 sequence sharing (p < 0.0001). Discussion: We have developed two unique classes of murine bladder cancer lines, UPPL and BBN, with gene expression and TIL profiles that closely correlate with human luminal and basal bladder cancers, respectively. The BBN and UPPL subtype specific models can serve as a tool for elucidating bladder cancer responses to immunotherapy. The mixed response of BBN963 tumors to PD-1 blockade should be an asset for assessing pathways mediating response to checkpoint blockade as well as the value of combination therapy. [C.S., R.S, B.V, W.K contributed equally to this work] Citation Format: Christof C. Smith, Ryoichi Saito, Lisa M. Bixby, Takanobu Utsumi, Jordan Kardos, Shengjie Chai, Sara E. Wobker, Bhavani Krishnan, Jeffrey S. Damrauer, Jonathan S. Serody, David Darr, Benjamin G. Vincent, William Y. Kim. Development of subtype specific mouse models of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2017-1654

Abstract 2174: Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML

Abstract Inhibition of oncogenic transcriptional programs is recognized to be a promising therapeutic strategy. SEL120-34A is a novel inhibitor of Cyclin- dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. SEL120-34A interacts with the ATP binding site of CDK8 in type I inhibitor fashion and forms several types of interactions, including halogen bonds with the protein’s hinge region and hydrophobic complementarities within its front pocket. Although the compound was only modestly active in solid tumor cell lines, it repressed phosphorylation of STAT5 Ser726 and could differentially inhibit viability of AML and ALL cell lines in vitro and in vivo, along with other type I CDK8 inhibitors. Transcriptomic analysis identified major transcriptional programs altered in responder cell lines, which strongly indicated that apart from repression of survival pathways, CDK8 inhibitors could induce differentiation in cell lines with leukemia stem cells characteristics. Further studies on a large panels of responder and non-responder cell lines identified robust biomarkers which could be used with high confidence for stratification and personalized approach in CDK8-dependent AML cases. Favorable pharmacokinetics, confirmed safety and in vivo efficacy in leukemia models provide the rationale for further clinical development of SEL120-34A. Citation Format: Tomasz Rzymski, Michal Mikula, Eliza Zylkiewicz, Agnieszka Dreas, Katarzyna Wiklik, Aniela Golas, Katarzyna Wojcik, Magdalena Masiejczyk, Iga Dudzicz, Katarzyna Kucwaj, Malgorzata Statkiewicz, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Jerzy Ostrowski, Urszula Kukliniska, Krzysztof Brzozka. Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2174. doi:10.1158/1538-7445.AM2017-2174

A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing.

The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.

Abstract 3226: Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models

Abstract PD-1 - PD-L1 pathway disruptors (e.g. nivolumab, pembrolizumab) are now being approved for treatment of patients with locally evolved or metastatic melanoma and advanced non-small cell lung cancer (NSCLC) who have progressed after first line platinum-based chemotherapy. These drugs have also antitumor efficacy in other tumor types (renal cell carcinoma, bladder, Hodgkin lymphoma, colorectal carcinoma with mismatch-repair deficiency ⋯). However, there is still needs to identify predictive biomarkers of response in order to select patients who will benefit from treatments. PD-L1 expression, mutation numbers, or mismatch repair (MMR) deficiencies were already demonstrated to influence response to PD-1 targeting therapies. However, they did not completely discriminate responders from non-responders or cut off levels are not yet precisely defined. Syngenic models are one of the only experimental models to evaluate immune system targeting therapies. Eight mouse models (4T1, A20, B16-F10, CT-26, EMT-6, MBT-2, LLC and Renca) were tested for response to PD-1 or PD-L1 targeting antibodies. From them, 3 models were characterized as non-responder (i.e. 4T1, LLC and RenCa) while other 7 models were showing to be sensitive to PD-1 inhibitors. PD-L1 expression (using RT-qPCR, WB and flow cytometry) was analyzed on cell lines at baseline and after 24h incubation with IFNg. At baseline, there is no significant difference between responders and non-responders whatever the level of analysis (mRNA, total PD-L1 protein expression or cell surface PD-L1 expression). However, after incubation with IFNg, there is a trend to segregate between responders and non-responders. Increased level of PD-L1 at the cell surface was evidenced for responders. IFNg signaling pathway used interferon regulatory factors (IRFs). IRF1 have been demonstrated to be responsible for both constitutive PD-L1 expression and early induction of PD-L1 after IFNg stimulation. mRNA expression of IRF1, IRF3, IRF7 and IRF9 were analyzed at baseline and after 24h incubation with IFNg. At baseline or after IFNg stimulation, there is no significant difference for IRF3, IRF7 and IRF9 mRNA expression between responders and non-responders. However, IRF1 mRNA is less expressed at baseline in responders as compared to non-responders. Moreover, increase in IRF1 expression after IFNg stimulation is higher in responders when compared to non-responders, which correlates well with changes in PD-L1 cell surface expression after IFNg stimulation. The total number of genomic variations was also analyzed using whole exome sequencing. Responder cell lines have highest number of variations in comparison to non-responder cell lines. In depth characterization of these syngeneic models will increase the understanding of how translatable are the results acquired from preclinical mouse studies. Citation Format: Anais Lagrange, Romain Boidot, Marc Hillairet de Boisferon, Olivier Duchamp, Jean-François Mirjolet, François Ghiringhelli. Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3226.

Targeting microRNAs: Towards a new tailored therapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a significant unmet medical need with very limited therapeutic options available. Although microRNA-21 (miR-21) has been shown to be upregulated in HCC, its contribution as an onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21, inhibited survival of a large subset of HCC cell lines. Sensitive HCC cell lines showed dose- and time-dependent induction of caspase 3/7 activity upon treatment with anti-miR-21. In contrast, non-responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. To better understand the consequences of miR-21 suppression in HCC, we carried out global gene expression profiling of anti-miR-21 treated sensitive liver cancer cells. Striking enrichment in miR-21 targets was noted among upregulated transcripts. Key cellular processes affected by miR-21 inhibition, including deregulation of metabolic pathways, were identified by gene ontology analysis. In summary, our data suggest that inhibition of miR-21 merits further investigation in the treatment of hepatocellular carcinoma.

Abstract 4787: Targeting miR-21 in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) remains a significant unmet medical need with few therapeutic options available. Micro RNA 21 (miR-21) has been shown to be upregulated in HCC, however, contribution of this onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21 (ANKRD46, DDAH1, RECK1) and inhibited survival of a large subset of HCC cell lines. Treatment of these sensitive HCC cell lines with anti-miR-21 resulted in dose- and time-dependent induction of caspase 3/7 activity. In contrast, non-responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. Further analysis of responder cell lines revealed robust induction of cell death, inhibition of cell migration and suppression of clonogenic growth upon treatment with miR-21 inhibitor. To better understand the consequences of miR-21 suppression in HCC, we carried out global gene expression profiling of anti-miR-21 treated sensitive liver cancer cells. Striking enrichment in miR-21 targets was noted among upregulated transcripts. Gene ontology analysis identified key cellular processes affected by miR-21 inhibition, including deregulation of metabolic pathways. In addition to the induction of direct miR-21 targets, cyclin H was found to be significantly downregulated upon miR-21 inhibition in the majority of responder cell lines. We hypothesize that inhibition of cyclin H expression, while an indirect effect of miR-21 suppression, could contribute to the activity of anti-miR-21 compounds. In summary, our data suggest that inhibition of miR-21 merits further investigation in the treatment of hepatocellular carcinoma. Citation Format: Sonya Zabludoff, Timothy Wagenaar, Francisco Adrian, Charles Allerson, Heike Arlt, Raffaele Baffa, Bal Bhat, Hui Cao, Scott Davis, Carlos Garcia-Echeverria, Kathrin Heermeier, Shih-Min Huang, Lan Jiang, Eric Marcusson, Christiane Metz-Weidmann, Adam Pavlicek, Jack Pollard, Jennifer Rocnik, Sabine Scheidler, Chaomei Shi, Fangxian Sun, Tatiana Tolstykh, Qunyan Yu, Gang Zheng, Dmitri Wiederschain. Targeting miR-21 in hepatocellular 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 4787. doi:10.1158/1538-7445.AM2014-4787

A Real-time Impedance-Based Screening Assay for Drug-Induced Vascular Leakage

Vascular leakage is a serious side effect of therapies based on monoclonal antibodies or cytokines which may lead to life-threatening situations. With the steady increase of new drug development programs for large molecules, there is an urgent need for reliable tools to assess this potential liability of new medicines in a rapid and cost-effective manner. Using human umbilical vein endothelial cells (HUVECs) as a model for endothelium, we established an impedance-based assay measuring the integrity of the endothelial cell monolayer in real time. We could demonstrate that the HUVEC monolayer in our system was a relevant model as cells expressed major junctional proteins known to be responsible for maintaining tightness as well as receptors targeted by molecules known to induce vascular leakage in vivo. We assessed the time-dependent loss of barrier function using impedance and confirmed that signals obtained corresponded well to those from standard transwell assays. We assayed a series of reference molecules which led to the expected change of barrier integrity. A nonspecific cytotoxic effect could be excluded by using human fibroblasts as a nonresponder cell line. Finally, we could show reversibility of vascular permeability induced by histamine, IL-1β, or TNF-α by coincubation with established antagonists, further demonstrating relevance of this new model. Taken together, our results suggest that impedance in combination with HUVECs as a specific model can be applied to assess clinically relevant vascular leakage on an in vitro level.

Abstract 695: The generation of a mono-selective CDK7 inhibitor.

Abstract Cyclin-depending kinase (CDK) family members that trigger passage through the cell cycle have been considered as attractive cancer targets for at least two decdes Yet, the first generation of pan-specific CDK inhibitors failed to show clinical efficacy due to their small therapeutic window. CDK family members that control processes such as transcription have caught less attention, although experimental evidence for an involvement in different pathological processes is emerging. CDK7 (or CAK) has essential roles in both the cell-division cycle and transcription, and serves as a direct link between the regulation of basal transcription and the cell cycle. In addition to its role as a transcription activator, CDK7 has been reported to act on specific transcription events by phosphorylating a series of specific transcription factors, such as the estrogen receptor and p53. These findings indicate that CDK7 is a potential target for cancer therapy, and perhaps inflammation. The detailed molecular characterization of the biological role of CDK7, as well as its therapeutic validation, is dependent on the development of highly specific and potent CDK7 inhibitors. We have developed a series of CDK7 inhibitors with picomolar potency on CDK7, more than 1000-fold selectivity over any other CDK, and single digit nM inhibition of sensitive cancer cell lines. Interestingly, a broad screen of >200 different tumor cell lines lead to the identification of responder and non-responder cell lines. Non-responder tumor lines, similar to primary cells are at least by a factor of 1000 less sensitive to the inhibition with the mono-selective CDK7 inhibitor. Our findings further indicate that CDK7 is neither essential for general transcription, nor for the progression through the cell cycle. It rather coordinates cell line specific biological processes. The effects of selective CDK7 inhibitors on tumor cell lines are clearly distinct from existing, nonselective pan-CDK inhibitors. The responder status for a subset of cancer cell lines can be predicted with a distinct biomarker. Chemical optimization of the selective inhibitor lead to a compound which shows dose-dependent pharmacological efficacy in mouse xenograft models after oral administration, with no signs of toxicity at the efficacious doses. Citation Format: Jan E. Eickhoff, Gunther Zischinsky, Axel Choidas, Peter Habenberger, Alexander Wolf, Carsten Degenhardt, Matthias Baumann, Anke Unger, Ahmad Ghallab, Doreen Werchau, Jan Hengstler, Peter Nussbaumer, Bert M. Klebl. The generation of a mono-selective CDK7 inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 695. doi:10.1158/1538-7445.AM2013-695

High tumor levels of IL6 and IL8 abrogate preclinical efficacy of the γ-secretase inhibitor, RO4929097

Interest continues to build around the early application of patient selection markers to prospectively identify patients likely to show clinical benefit from cancer therapies. Hypothesis generation and clinical strategies often begin at the preclinical stage where responder and nonresponder tumor cell lines are first identified and characterized. In the present study, we investigate the drivers of in vivo resistance to the γ-secretase inhibitor RO4929097. Beginning at the tissue culture level, we identified apparent IL6 and IL8 expression differences that characterized tumor cell line response to RO4929097. We validated this molecular signature at the preclinical efficacy level identifying additional xenograft models resistant to the in vivo effects of RO4929097. Our data suggest that for IL6 and IL8 overexpressing tumors, RO4929097 no longer impacts angiogenesis or the infiltration of tumor associated fibroblasts. These preclinical data provide a rationale for preselecting patients possessing low levels of IL6 and IL8 prior to RO4929097 dosing. Extending this hypothesis into the clinic, we monitored patient IL6 and IL8 serum levels prior to dosing with RO4929097 during Phase I. Interestingly, the small group of patients deriving some type of clinical benefit from RO4929097 presented with low baseline levels of IL6 and IL8. Our data support the continued investigation of this patient selection marker for RO4929097 and other types of Notch inhibitors undergoing early clinical evaluation.

Gene Expression Profiles for Predicting the Efficacy of the Anticancer Drug 5-Fluorouracil in Breast Cancer

Chemotherapy is an important postsurgery adjuvant therapy in the treatment of breast cancer. However, because of the individual genotype differences of patients, the drug efficacy differs from person to person, even when the same chemotherapy drug is administered. The purpose of this research was to probe the gene expression profiles to predict the efficacy of 5-fluorouracil (5-FU), the common drug used in chemotherapy for various type of cancers, in Taiwanese breast cancer patients. Microarray analysis was conducted on the cancer cell line ZR-75-1 with and without 5-FU stimulation to identify the differentially expressed genes. The significant overexpressed gene groups were selected after bioinformatics software analysis to explore the molecular mechanism of 5-FU. Six strains of breast cancer cell line purchased from American Type Culture Collection were used to analyze the expression profiles of the above target gene groups. IL18, CCL28, CXCL2, SOD1, HRAS, FDXR, and CHI3L1 genes were significantly differentially expressed in 5-FU responder and nonresponder cell lines. The selected gene groups were validated with 20 strains of breast cancer primary cultures established previously in our laboratory. The experimental results demonstrated that FAM46A, IL18, CCL28, TNF, CXCL2, PLEKHA8, HRAS, FDXR, and CHI3L1 genes showed statistically significant differential expression between primary breast cancer culture cells that respond and nonrespond to 5-FU. Six genes, IL18, CCL28, CXCL2, HRAS, FDXR, and CHI3L1, showed significant differential expression pattern in both American Type Culture Collection and primary breast cancer cultured cells. The findings of this study may serve as basis for predicting the effectiveness of 5-FU on breast cancer.

Altered Cellular Protein Levels of Tumor Suppressor Genes and Heat Shock Elements (TRAP1) Indicate Sensitivity to the Proteasome Inhibitor Bortezomib (Velcade®) in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a distinct lymphatic neoplasia characterized by a poor clinical outcome with only transient response to conventional chemotherapy. In various phase II studies the proteasome inhibitor Bortezomib (Velcade®) has demonstrated a high clinical efficacy with up to 60% remission rates in relapsed MCL, however, the target pathways of proteasome inhibition remain largely unknown. Additionally, as the mode of action involves proteasome mediated protein degradation, only analysis of the protein level may reliably identify the cellular signal pathways involved. Four MCL cell lines (HBL-2, Granta 519, Jeko-1, NCEB-1) were exposed to Bortezomib at the minimal cytotoxic concentration (25nmol) and harvested for analysis at 0, 1, 4 and 8 hours of exposure. To assess the early alterations of cellular protein levels induced by Bortezomib-effected proteasome inhibition, the whole cellular proteome was screened applying two-dimensional gel electrophoresis. A high percentage of proteins were initially affected by proteasome inhibition, increasing their cellular content. Analyzing triplicate experiments, approximately 20 % of all cellular proteins detected on the 2D-gels (201/1013) exhibited an alteration of cellular protein content by a factor of >5, while 14 % (148) of the proteins were altered by >10 x. As expected about 79 % (158/201) of these proteins showed an increase of cellular protein, and only 21 % (44/201) of the proteins were downregulated. Approximately 65 % (133/201) of these proteins were already dysregulated after 1 hour whereas the 68 remaining proteins reached the threshold level after 4 hours. Cross cell line comparison yielded 41 reproducibly altered proteins in 3 responder cell lines. Among these proteins are (putative) tumor suppressors (ENO-1, fumarate hydratase), heat shock response elements (TRAP1, HSPA8, HSP72) and structural proteins (centrosomal protein CEP290). In the non-responder cell line (NCEB-1), expression of only 22 of these 41 proteins was altered after exposure to Bortezomib. These unspecific proteins involved some heat shock response elements (HSPA8, HSP72), the respiratory chain (NADH-ubiquinon oxidoreductase 24kDa mitochondrial precursor) and signal transduction (LSP1). In contrast, the cellular protein levels of the putative tumor suppressor genes, as well as the actin cross-linking protein alpha-fodrin were altered only in the responder cell lines. RNA-expression analysis detected highly diverging expression patterns after Bortezomib treatment in all four cell lines. No down-regulation of proliferation associated genes was detected, and the expression of heat shock response related genes were increased. As predicted, due to its mode of action, there was no correlation between the detected alterations of cellular protein levels and RNA-expression after Bortezomib treatment. In summary, Bortezomib treatment specifically affected the cellular protein levels of tumor suppressor genes and distinct heat shock response elements (TRAP1) in sensitive cell lines. Currently primary patient samples are being analyzed to confirm the predictive value of these molecular markers.

Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields

The issue of adverse health effects of extremely low-frequency electromagnetic fields (ELF-EMFs) is highly controversial. Contradictory results regarding the genotoxic potential of ELF-EMF have been reported in the literature. To test whether this controversy might reflect differences between the cellular targets examined we exposed cultured cells derived from different tissues to an intermittent ELF-EMF (50Hz sinusoidal, 1mT) for 1–24h. The alkaline and neutral comet assays were used to assess ELF-EMF-induced DNA strand breaks. We could identify three responder (human fibroblasts, human melanocytes, rat granulosa cells) and three non-responder cell types (human lymphocytes, human monocytes, human skeletal muscle cells), which points to the significance of the cell system used when investigating genotoxic effects of ELF-EMF.

Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary--K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2(C95Y), functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.

Defect in Th1-Like Cells of Nonresponders to Hepatitis B Vaccine

ABSTRACT: Peripheral blood lymphocytes from nonresponders to hepatitis B vaccine (HBsAg) failed to undergo a proliferative response to recombinant HBsAg in vitro, whereas cells from responders proliferated vigorously. The lack of proliferative response was not due to defective antigen presentation in that MHC-identical responder and nonresponder antigen presenting cells were equally effective in stimulating responder T cells. Nonresponder T cells did not proliferate in response to antigen-pulsed MHC identical responder antigen presenting cells. The present study demonstrated that: 1) there were no detectable (1 in <20 × 10 4) HBsAg-precursor T cells in any of the nonresponders, while in responders the frequency of HBsAg-precursor T cells ranged from 1 in 3.2 × 10 3 to 1 in 40 × 10 3; 2) nonresponder cell cultures did not secrete IL-2 in response to HBsAg stimulation; 3) exogenous recombinant IL-2 did not restore the proliferative response of the T cells in HBsAg-pulsed cultures of nonresponders. These results suggest that the cellular basis for the lack of response to HBsAg is a defect in HBsAg-specific Th1-like cells; either there is an absence of the Th1 cells or cells with TCR specificity for HBsAg are present but are unresponsive to the HBsAg peptide-MHC complex ( i.e., anergy or tolerance).