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Supplementary Data from Interactions of the Hdm2/p53 and Proteasome Pathways May Enhance the Antitumor Activity of Bortezomib

Purpose: p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM).Experimental Design: We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3.Results: The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells.Conclusions: This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy. (Clin Cancer Res 2009;15(23):7153–60)

Understanding the biology of CHO cells at a cellular level is extremely valuable to the pharmaceutical industry, as it presents the opportunity to rationally engineer clonally derived cell lines (CDCLs) with elevated recombinant specific productivity (Qp). To date, relatively little is understood about the intracellular pathways which influence desirable phenotypes in CHO cell lines. In this study we identified phosphoproteins and total proteins which are differentially expressed between high and low Qp IgG4 producing industry CHO cell lines, some of which upon further investigation, could potentially be used as targets to engineer high Qp CDCLs. We highlighted the importance of phosphoproteins and total proteins associated with amino acid sensing in high Qp cell lines. We also identified increased expression of proteins which promote progression through the cell cycle in low Qp cell lines, suggesting that lower Qp CDCLs are focusing their translational machinery on translating cell cycle associated mRNAs.

The Caco-2 cell line is composed of a heterogeneous mix of cells; isolation of individual clonal populations from this mix allows for specific mechanisms and phenotypes to be further explored. Previously we exposed Caco-2 cells to inorganic copper sulphate or organic copper proteinate to generate resistant variant populations. Here we describe the isolation and characterisation of clonal subpopulations from these resistant variants to organic (clone Or1, Or2, Or3) or inorganic (clone In1 and In2) copper. The clones show considerable homogeneity in response to Cu-induced toxicity and heterogeneity in morphology with variations in level of cross-resistance to other metals and doxorubicin. Population growth was reduced for Cu-resistant clones In2 and Or3 in selective pressure relative to parental Caco-2 cells. Gene expression analysis identified 4026 total (2102 unique and 1924 shared) differentially expressed genes including those involved in the MAP Kinase and Rap1 signalling pathways, and in the focal adhesion and ECM-receptor contact pathways. Gene expression changes common to all clones included upregulation of ANXA13 and GPx2. Our analysis additionally identified differential expression of multiple genes specific to copper proteinate exposure (including overexpressed UPK1B) in isolated clones Or1, Or2 and Or3 and CuSO4 exposure (including decreased AIFM2 expression) in isolated clones In1 and In2. The adaptive transcriptional responses established in this study indicate a cohort of genes, which may be involved in copper resistance regulation and chronic copper exposure.

Pancreatic cancer is usually advanced and drug resistant at diagnosis. A potential therapeutic approach outlined here uses nanoparticle (NP)-based drug carriers, which have unique properties that enhance intra-tumor drug exposure and reduce systemic toxicity of encapsulated drugs. Here we report that patients whose pancreatic cancers express elevated levels of Death Receptor 5 (DR5) and its downstream regulators/effectors FLIP, Caspase-8, and FADD had particularly poor prognoses. To take advantage of elevated expression of this pathway, we designed drug-loaded NPs with a surface-conjugated αDR5 antibody (AMG 655). Binding and clustering of the DR5 is a prerequisite for efficient apoptosis initiation, and the αDR5-NPs were indeed found to activate apoptosis in multiple pancreatic cancer models, whereas the free antibody did not. The extent of apoptosis induced by αDR5-NPs was enhanced by down-regulating FLIP, a key modulator of death receptor- mediated activation of caspase-8. Moreover, the DNA topoisomerase-1 inhibitor camptothecin (CPT) down-regulated FLIP in pancreatic cancer models and enhanced apoptosis induced by αDR5-NPs. CPT-loaded αDR5-NPs significantly increased apoptosis and decreased cell viability in vitro in a caspase-8- and FADD-dependent manner consistent with their expected mechanism-of-action. Importantly, CPT-loaded αDR5-NPs markedly reduced tumor growth rates in vivo in established pan- creatic tumor models, inducing regressions in one model. These proof-of-concept studies indicate that αDR5-NPs loaded with agents that downregulate or inhibit FLIP are promising candidate agents for the treatment of pancreatic cancer.

To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3 days showed that cessation of growth, associated with increased G1/S transition and inhibition of M/G1 transition was accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1).Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting not only our ability to study CHO cell biology but also detect host cell protein (HCP) contaminants in the final mAb drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of four commercial mAb drug products using the extended protein sequence database revealed the presence of microprotein impurities for the first time. We also show that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation as well as the regulation of protein synthesis in CHO cell lines.

Background The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. Results We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. Conclusion This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

Objectives We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. Results Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. Conclusion These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.

Chinese hamster ovary (CHO) cells are one of the most commonly used host cell lines used for the production human therapeutic proteins. Much research over the past two decades has focussed on improving the growth, titre and cell specific productivity of CHO cells and in turn lowering the costs associated with production of recombinant proteins. CHO cell engineering has become of particular interest in recent years following the publication of the CHO cell genome and the availability of data relating to the proteome, transcriptome and metabolome of CHO cells. However, data relating to the cellular post-translational modification (PTMs) which can affect the functionality of CHO cellular proteins has only begun to be presented in recent years. PTMs are important to many cellular processes and can further alter proteins by increasing the complexity of proteins and their interactions. In this review, we describe the research presented from CHO cells to date related on three of the most important PTMs; glycosylation, phosphorylation and ubiquitination.

MicroRNAs are increasingly being used to enhance relevant pathways of interest during CHO cell line development and to optimise biopharmaceutical production processes. Previous studies have demonstrated that genetic manipulation of microRNAs has led to the development of highly productive phenotypes by increasing cell density through modifying the cell cycle, extending the culture lifespan by delaying apoptotic mechanisms, or improving the energetic flux by targeting mitochondrial metabolism. Re-programming mitochondrial metabolism has arisen as a potential area of interest due to the potential to decrease the Warburg effect and increase cell specific productivity with significant impact on the manufacture of recombinant therapeutic proteins. In this study, we have demonstrated a role for miR-31* to enhance specific productivity in CHO cells by boosting oxidative phosphorylation in the mitochondria. A detailed analysis of the mitochondrial metabolism revealed that miR-31* transfection increases basal oxygen consumption and spare respiratory capacity that leads to an increase in ATP production. Additionally, a proteomic analysis unveiled a number of potential targets involved in fatty acid metabolism and the TCA cycle, both implicated in mitochondrial metabolism. This data demonstrates a potential role for miR-31* to reprogramme the mitochondrial energetic metabolism and increase recombinant protein production in CHO cells.

The modern world has seen exposure of bacterial communities to toxic metals at selective levels. This manifests itself both intentionally, through medicines and un-intentionally through waste streams. There is growing concern that selective exposure to metals may be linked to microbial resistance to antibiotics. For a microbe to become resistant to a specific metal it must first come in contact with it. The transition metal copper has the ability to enter bacterial cells without need for a copper specific uptake mechanism. Copper is commonly used as an antimicrobial in the healthcare industry, consumer products and as a growth promoter of livestock in the agricultural sector. Here we report a study into the uptake of different organic and inorganic sources of copper. A whole-cell bacterial biosensor was developed to quantify the specific uptake of copper from various sources. Furthermore, a cell-free sensor was utilized to investigate the response to copper sources when uptake is eliminated as a factor. The data within suggest inorganic copper to have greatly reduced uptake compared to organic sources and that there is significant difference between copper oxides, Cu2O and CuO.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp) rising from 105 to 107 m−1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine–cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

Pancreatic cancer remains among the most lethal cancers worldwide, with poor early detection rates and poor survival rates. Patient-derived xenograft (PDX) models have increasingly been used in preclinical and clinical research of solid cancers to fulfil unmet need. Fresh tumour samples from human pancreatic adenocarcinoma patients were implanted in severe combined immunodeficiency (SCID) mice. Samples from 78% of treatment-na&iuml, ve pancreatic ductal adenocarcinoma patients grew as PDX tumours and were confirmed by histopathology. Frozen samples from F1 PDX tumours could be later successfully passaged in SCID mice to F2 PDX tumours. The human origin of the PDX was confirmed using human-specific antibodies, however, the stromal component was replaced by murine cells. Cell lines were successfully developed from three PDX tumours. RNA was extracted from eight PDX tumours and where possible, corresponding primary tumour (T) and adjacent normal tissues (N). mRNA profiles of tumour vs. F1 PDX and normal vs. tumour were compared by Affymetrix microarray analysis. Differential gene expression showed over 5000 genes changed across the N vs. T and T vs. PDX samples. Gene ontology analysis of a subset of genes demonstrated genes upregulated in normal vs. tumour vs. PDX were linked with cell cycle, cycles cell process and mitotic cell cycle. Amongst the mRNA candidates elevated in the PDX and tumour vs. normal were SERPINB5, FERMT1, AGR2, SLC6A14 and TOP2A. These genes have been associated with growth, proliferation, invasion and metastasis in pancreatic cancer previously. Cumulatively, this demonstrates the applicability of PDX models and transcriptomic array to identify genes associated with growth and proliferation of pancreatic cancer.

Product degradation, such as clipping, is a common quality issue in the production of Fc-fusion proteins from Chinese hamster ovary (CHO) cells. Degradation of proteins is mainly due to the action of either intracellular or extracellular host cell proteases. This study was carried out to understand more fundamentally the intracellular events that may play a role in determining why cell lines from the same cell line development project can vary with regards to the extent of Fc-fusion protein clipping. The cell lines that displayed the highest levels of clipping also produced less product than the cell lines with a lower level of clipping. In this study we applied differential quantitative label-free LC-MS/MS proteomic analysis to group clonally-derived cell lines (CDCLs) based on the level of clipping of the Fc-fusion protein. The analysis was carried out over two times points in culture and clones were designated as either having 'high' or 'low' clipping phenotypes. We have identified 200 differentially expressed proteins using quantitative label-free LC-MS/MS analysis between the two experimental groups. Functional assessment of the resultant proteomic data using Gene Ontology analysis showed a significant enrichment of biological processes and molecular functions related to protein folding, response to unfolded protein and protein translation. The levels of several proteases were also increased. This study identified protein targets that could be modified using cell line engineering approaches to improve the quality of recombinant Fc-fusion protein production in the biopharmaceutical industry.

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes, however alternative mRNA splicing, has thus far, received little attention. In this study, we utilised RNASeq for transcriptomic analysis of a monoclonal antibody producing CHOK1 cell line subjected to a temperature shift. More than 2,365 instances of differential splicing were observed 24hrs after the reduction of cell culture temperature. 1,163 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using qPCR in the monoclonal antibody producing CHOK1 line used for RNASeq and a further 2 CHOK1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilised to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

Background Multiple myeloma (MM) is a complex heterogeneous disease. Various risk stratification models have been recommended including cytogenetic and FISH analysis to identify high-risk patients who may benefit from novel treatments, but such facilities are not widely available. The International Scoring System (ISS) using beta-2-microglobulin and albumin remains a widely used prognostic scoring system in many clinical practices; however it is not useful in predicting response to treatment in MM. The aim of this study is to identify clinically useful biomarkers to predict response to treatment containing bortezomib. Methods 17 MM patient serum samples (9 responders/8 non-responders) were used for the discovery phase (label-free mass spectrometry) and an additional 20 MM patient serum samples were used for the ELISA-based validation phase (14 responders/6 non-responders). Results CLU and ANG mean levels were higher in the responders group, while Complement C1q had lower concentrations. The combination of all standard biomarkers (albumin, beta-2-microglobulin (ß2M), paraprotein and kappa/lambda (K/L) ratio) had an AUC value of 0.71 with 65% correct classification, while an overall combination of new candidate protein biomarkers with standard biomarkers had an AUC value of 0.89 with 85.3% correct classification. Conclusions A combination of new and standard biomarkers consisting of CLU, ANG, C1Q, albumin, ß2M, paraprotein and K/L ratio may have potential as a novel panel of biomarkers to predict MM response to treatment containing bortezomib. General significance Use of this biomarker panel could facilitate a more personalized therapy approach and to minimize unnecessary side effects from ineffective drugs., Highlights • CLU and ANG mean levels were higher in the responders group. • Complement C1q mean levels were lower in the responders group. • Standard biomarker panel had an AUC value of 0.708 with 64.9% correct classification. • New biomarker panel had an AUC value of 0.89 with 85.3% correct classification. • Use of this biomarker panel could facilitate a more personalized therapy approach.

Pancreatic cancer is one of the most fatal human cancers with a 5-year survival rate of approximately 6%. Pancreatic Ductal Adenocarcinoma (PDAC) is the predominant (~90%) histological type among pancreatic cancers. The poor prognosis of PDAC is due to a combination of factors including late stage diagnosis at a locally advanced or metastatic stage, due to its asymptomatic progression. PDAC is a highly chemoresistant malignancy owing to its genetic heterogeneity and dense stromal environment. FOLFIRINOX (FFX) is a combinatorial chemotherapeutic approach consisting of 5-Fluorouracil (5-FU)/Leucovorin (LV), Irinotecan and Oxaliplatin, and is a first-line treatment option for pancreatic cancer patients. Despite its superior clinical benefit for the treatment of metastatic PDAC compared to gemcitabine, its high toxicity profile limits its use to patients with high performance status. In-depth pharmacodynamic (PD) modelling of the interactions between the individual agents of the regimen is required. Here, the effect of the FFX regime was investigated using both established and primary pancreatic cancer cell-lines cultured as 2D and 3D models. Cells were exposed to a concentration range of each agent in single, double and triple combinations. Cell viability assayed after 5 days. The addition of LV to 5-FU had no effect in vitro in Panc-1, Mia-PaCa-2 and primary PT99 cells, and was excluded from further analysis. The active metabolite of Irinotecan, SN-38, was used in-vitro. The Bliss Independent method was used to fit the cell viability data for all cell lines using SAS 9.4 software. A psi value less than 1 suggests synergistic interactions. Of 4 cell lines tested, MIA-PaCa-2 cells were the most sensitive to the agents of the FFX regimen, with the triple combination demonstrating synergistic interactions with a psi value of 0.036. However, in Panc-1, BxPC-3 and PT127 cells the triple combination was antagonistic, with psi values of 5.167, 3.598 and 5.349 respectively. For the double combination, Ox + SN-38 was shown to have synergistic interactions in 3 of 4 cell lines. The 5-FU + SN-38 and 5-FU + Ox combinations were synergistic in 3 of 4 cell lines. The differences in cellular response observed between the cell lines mirrors the heterogeneity of patients in the clinic and highlights the importance of mathematical modelling to better understand the PD interactions of multidrug treatment regimens. While the triple combinations were determined as antagonistic, meaning the net effect is less than the additive effect of individual agents, it still resulted in greater cell kill than any agent alone. Therefore despite findings of antagonism this further supports the need for all 3 drugs. In conclusion, the results suggest a complex interplay between the three agents, and the PD data could be used to assess whether lower doses and/or alternate dosing schedules would be feasible. This potentially translatable finding may result in FFX being available to more PDAC patients. Citation Format: Taylor Allen-Coyle, Jin Niu, Eva Welsch, Fiona O'Neill, Martin Clynes, Finbarr O'Sullivan, Donald E. Mager, Robert M. Straubinger, Sandra Roche. Could pharmacodynamic modelling of FOLFIRINOX lead to more therapeutically advantageous treatment of pancreatic cancer? [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-028.

Pancreatic cancer survival rate is poor, with a 5-year survival rate of 7% in the Republic of Ireland. Over the last 40 years, pancreatic cancer is one of the only solid tumor types to have minimal improvement in patient outcome. In Ireland, pancreatic cancer surgical resection is limited to two hospitals: Cork University Hospital and St Vincent's University Hospital (SVUH) in Dublin. In collaboration with SVUH, we have established Ireland's only pancreatic cancer patient-derived xenograft (PDX) biobank program. Tumor material from candidate patients following surgical resection was cold transferred and implanted subcutaneously into CB17/Icr-Prkdcscid mice. At time of implant and continuation of generations, snap frozen material was collected. Using miRNA and mRNA microarray technology, matched adjacent normal tissue, original tumor material and first generation (F1) PDX tumor material was interrogated. Differential expression analysis was carried out on all samples with comparisons of normal, tumor and F1 generation. Across all comparisons there were approximately 4000 genes and miRNAs found to be differential expressed. In focusing on genes that were upregulated in tumor samples compared to normal and further upregulated in the PDX F1 samples compared to the tumor samples, we identified 88 genes of interest. Biological processes such as cell division, mitotic cell cycle processes and cell cycle processes characterized a significant number of these genes. Two of the key genes of interest in this analysis were TSPAN1 and TPX2. TSPAN1 was upregulated 9.5-fold in tumor-normal comparison and upregulated a further 2.3-fold in the PDX F1 compared to the patient tumor. TPX2 was upregulated 4.4-fold in the tumor-normal comparison and a further 3.9-fold increase in the PDX tumor compared to patient tumor. TSPAN1 has previously been shown to increase the metastatic and invasive potential of pancreatic ductal adenocarcinoma cell lines. TPX2, a microtubule-associated protein, has been shown to reduce tumor growth in vivo when silenced. The pancreatic PDX biobank represents a versatile, expandable patient cohort for preclinical investigation. Analysis of gene expression profiles of normal vs tumor and tumor Vs PDX showed genes with associated tumor proliferation and aggressiveness. Citation Format: Sandra Roche, Fiona O'Neill, Niall Swan, Ninfa L. Straubinger, Neil T. Conlon, Jean Murphy, Kevin Conlon, Ray McDermott, Justine Meiller, Justin Geoghegan, Michael Moriarty, Robert M. Straubinger, Martin Clynes. Establishment and characterization by expression microarrays of a patient-derived xenograft biobank for human pancreatic adenocarcinoma [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 1678.

AIM To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. METHODS Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward’s clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. RESULTS Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid and cholesterol metabolic processes, small molecule transport and a range of responses to external stimuli, while similar analysis of the DE protein list identified gene expression/transcription, epigenetic mechanisms, DNA replication, differentiation and translation ontology categories. The DE protein and gene lists were found to share 15 biological processes including for example epithelial cell differentiation [P value ≤ 1.81613E-08 (protein list); P ≤ 0.000434311 (gene list)] and actin filament bundle assembly [P value ≤ 0.001582797 (protein list); P ≤ 0.002733714 (gene list)]. Analysis was conducted on the three data streams acquired in parallel to identify targets undergoing potential miRNA translational repression identified 34 proteins, whose respective mRNAs were detected but no change in expression was observed. Of these 34 proteins, 27 proteins downregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 19 unique anti-correlated/upregulated microRNAs and 7 proteins upregulated in the Caco-2 cell line relative to the HT-29 cell line and predicted to be targeted by 15 unique anti-correlated/downregulated microRNAs. CONCLUSION This first study providing “tri-omics” analysis of the principal intestinal cell line models Caco-2 and HT-29 has identified 34 proteins potentially undergoing miRNA translational repression.

Background: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. Methods: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry. The potential anti-tumour effects of MAb 9E1 were investigated in vitro together with immunohistochemical analysis of the 9E1 target antigen in normal and cancer tissues. Results: MAb 9E1 significantly decreases invasion in pancreatic, lung squamous and breast cancer cells and silencing of its target antigen, which was revealed as AnxA6, leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro. IHC using MAb 9E1 revealed that AnxA6 exhibits a high prevalence of membrane immunoreactivity across aggressive tumour types with restricted expression observed in the majority of normal tissues. In pancreatic ductal adenocarcinoma, high AnxA6 IHC score correlated with the presence of tumour budding at the invasive front of tumours (P=0.082), the presence of perineural invasion (P=

Background Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion. Methodology PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n = 48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays. Findings Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion. Interpretation Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion., Highlights • This study explored neutrophil adhesion in cystic fibrosis. • Altered membrane cholesterol lead to increased adhesion. • Circulating inflammatory mediators caused increased calpain activity and reduced membrane cholesterol content. In patients with cystic fibrosis (CF), chronic inflammation in the circulation, in part originating from the pulmonary compartment, leads to decreased membrane cholesterol in circulating neutrophils, resulting in increased cell adhesion. The mechanism of action involves proteolytic down-regulation of the cholesterol trafficking protein caveolin-1. The overall effect of lung transplant therapy, or CFTR potentiator treatment, was to significantly diminish the circulating inflammatory burden thereby permitting caveolin-1 expression, with concomitant decreased CF cell adhesion and significant clinical improvement.

Background Lung cancer is the leading cause of cancer-related mortality in both men and women throughout the world. The need to detect lung cancer at an early, potentially curable stage, is essential and may reduce mortality by 20%. The aim of this study was to identify distinct proteomic profiles in bronchoalveolar fluid (BALF) and plasma that are able to discriminate individuals with benign disease from those with non-small cell lung cancer (NSCLC). Methods Using label-free mass spectrometry analysis of BALF during discovery-phase analysis, a significant number of proteins were found to have different abundance levels when comparing control to adenocarcinoma (AD) or squamous cell lung carcinoma (SqCC). Validation of candidate biomarkers identified in BALF was performed in a larger cohort of plasma samples by detection with enzyme-linked immunoassay. Results Four proteins (Cystatin-C, TIMP-1, Lipocalin-2 and HSP70/HSPA1A) were selected as a representative group from discovery phase mass spectrometry BALF analysis. Plasma levels of TIMP-1, Lipocalin-2 and Cystatin-C were found to be significantly elevated in AD and SqCC compared to control. Conclusion The results presented in this study indicate that BALF is an important proximal biofluid for the discovery and identification of candidate lung cancer biomarkers. General significance There is good correlation between the trend of protein abundance levels in BALF and that of plasma which validates this approach to develop a blood biomarker to aid lung cancer diagnosis, particularly in the era of lung cancer screening. The protein signatures identified also provide insight into the molecular mechanisms associated with lung malignancy., Highlights • BALF proteome harbours significant differences between control and NCSLC. • Significant difference in proteins detected between AD and SqCC in BALF • Good correlation between proteins with altered abundance levels in BALF and plasma • Cystatin-C, TIP-1 and Lipocalin significantly changed in both BALF and plasma.

Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.

In order to study the impact of zinc and copper on the titer levels of mAb and recombinant protein in CHO cells, the IgG-expressing (DP12) and EPO-expressing (SK15) cell lines were cultured in chemically defined media with increasing concentrations of either metal. Supplementation with 25 mg/l in CDM media resulted in a significant increase in EPO (1.7-fold) and IgG (2.6-fold) titers compared to control (no added zinc). Titers at this Zn concentration in CDM containing the insulin replacing agent aurintricarboxylic acid (ATA) (CDM + A) showed a 1.8-fold (EPO) and 1.2-fold (IgG) titers increase compared to control. ATA appeared to also reduce the specific productivity (Qp) enhancement induced by Zn-25, with up to 4.9-fold (DP12) and 1.9-fold (SK15) Qp increase in CDM compared to the 1.6-fold (DP12) and 1.5-fold (SK15) Qp increase observed in CDM + A. A 31% reduced Viable Cell Density (VCD) in DP12 was observed in both Zn-supplemented media (3 × 10(6) cells/ml vs 4.2 × 10(6) cells/ml, day 5), whereas SK15 Zn-25 cultures displayed a 24% lower peak only in CDM + A (2.2 × 10(6) cells/ml vs 3.2 × 10(6) cells/ml, day 5). Supplementation with copper at 13.7–20 mg/l resulted in less significant cell line/product-type dependent effects on titer, VCD and Viability. Analysis of the energetic phenotype of both cell lines in 25 mg/l Zn-supplemented CDM media revealed a twofold increase in the oxygen consumption rate (OCR) compared to non-supplemented cells. Together, these data suggest that high zinc supplementation may induce an increase in oxidative respiration metabolism that results in increased Qp and titers in suspension CHO cultures. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10616-019-00334-1) contains supplementary material, which is available to authorized users.

Single‐use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO‐K1, and CHO‐DP12, cultured in media conditioned in an older single‐use bioreactor (SUB) film (F‐1) and a newer generation film (F‐2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F‐1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F‐2 film. Proteomic profiling of CHO cells cultured in the F‐1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F‐2 films when compared to F‐1 films including bDtBPP, potentially explaining improved CHO cell growth in F‐2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess. Science Foundation Ireland Irish Industry Technical Group (Allergan Pharmaceuticals Ireland, BioMarin Manufacturing Ireland Ltd., Eli Lilly and Company, Genzyme Ireland Ltd. A Sanofi Company, Janssen Biologics, MSD and Pfizer Ireland Pharmaceuticals) Dublin City University

Understanding the events at a protein level that govern the progression from melanoma in situ to invasive melanoma are important areas of current research to be developed. Recent advances in the analysis of formalin-fixed, paraffin-embedded tissue by proteomics, particularly using the filter-aided sample preparation protocol, has opened up the possibility of studying vast archives of clinical material and associated medical records. In the present study, quantitative protein profiling was performed using tandem mass spectrometry, and the proteome differences between melanoma in situ and invasive melanoma were compared. Biological pathway analyses revealed several signalling pathways differing between melanoma in situ and invasive melanoma, including metabolic pathways and the phosphoinositide 3-kinase-Akt signalling pathway. Selected proteins of interest (14–3-3ε and fatty acid synthase) were subsequently investigated using immunohistochemical analysis of tissue microarrays. Identifying the key proteins that play significant roles in the establishment of a more invasive phenotype in melanoma may ultimately aid diagnosis and treatment decisions.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide, it develops in a relatively symptom-free manner, leading to rapid disease progression and metastasis, leading to a 5-year survival rate of less than 5%. A lack of dependable diagnostic markers and rapid development of resistance to conventional therapies are among the problems associated with management of the disease. A better understanding of pancreatic tumour biology and discovery of new potential therapeutic targets are important goals in pancreatic cancer research. This study describes the comparative quantitative LC-MS/MS proteomic analysis of the membrane-enriched proteome of 10 human pancreatic ductal adenocarcinomas, 9 matched adjacent-normal pancreas and patient-derived xenografts (PDXs) in mice (10 at F1 generation and 10 F2). Quantitative label-free LC-MS/MS data analysis identified 129 proteins upregulated, and 109 downregulated, in PDAC, compared to adjacent-normal tissue. In this study, analysing peptide MS/MS data from the xenografts, great care was taken to distinguish species-specific peptides definitively derived from human sequences, or from mice, which could not be distinguished. The human-only peptides from the PDXs are of particular value, since only human tumour cells survive, and stromal cells are replaced during engraftment in the mouse, this list is, therefore, enriched in tumour-associated proteins, some of which might be potential therapeutic or diagnostic targets. Using human-specific sequences, 32 proteins were found to be upregulated, and 113 downregulated in PDX F1 tumours, compared to primary PDAC. Differential expression of CD55 between PDAC and normal pancreas, and expression across PDX generations, was confirmed by Western blotting. These data indicate the value of using PDX models in PDAC research. This study is the first comparative proteomic analysis of PDAC which employs PDX models to identify patient tumour cell-associated proteins, in an effort to find robust targets for therapeutic treatment of PDAC.

The proteomic data presented in this article provide supporting information to the related research article "Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of Ubiquitin Carboxyl-Terminal Hydrolase 14" (Costello et al., in press) [1]. Control and microRNA-378 depleted CHO DP12 cells were profiled using label-free quantitative proteomic profiling. CHO DP12 cells were collected on day 4 and 8 of batch culture, subcellular proteomic enrichment was performed, and subsequent fractions were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Here we provide the complete proteomic dataset of proteins significantly differentially expressed by greater than 1.25-fold change in abundance between control and miR-378 depleted CHO DP12 cells, and the lists of all identified proteins for each condition.

The impact of local structure on mRNA translation is not well-defined pertaining to the 5′ UTR. Reports suggest structural remodelling of the 5′ UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5′ UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5′ UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer. Science Foundation Ireland Update issue date during checkdate report - AC

miRNAs are potent molecular regulators of cellular behaviour. The manipulation of these small non-coding RNAs has been used to enhance industrially relevant phenotypes in Chinese Hamster Ovary (CHO) cells. We investigated the stable depletion of six miRNAs; miR-204-5p, 338-3p, 378-3p, 409-3p, 455-3p and 505-3p, robustly associated with cell growth rate from a previous profiling study. Inhibition of endogenous miR-378-3p function by miRNA-sponge-decoy improved peak cell density by 59%. Quantitative label free LC-MS/MS proteomic analysis of the fractionated cell cultures at day 4 and 8 of batch culture found 216 cytosolic and 114 membrane-associated proteins differentially expressed with stable miR-378-3p depletion. qRT-PCR of 8 genes; Clic4, Hnrnpa1, Prdx1, Actn4, Usp14, Srxn1, Canx and Gnb1, with unidirectional differential protein expression over the two time points of analysis was carried out. In-silico predictive algorithms; TargetScan and miRDB, were used to decipher possible direct targets of miR-378-3p. The Ubiquitin carboxyl-terminal hydrolase 14 (Usp14) protein was identified in the cytosolic fractions at both timepoints as differentially expressed with an increased abundance of 1.58-fold in the miR-378-3p depleted cells on day 8. Usp14 is a deubiquitinase (DUB) with previous reports of its up-regulation leading to increased proliferation of cancer cells. Overexpression of Usp14 in CHO cells had significant effects on cell growth supporting a role for Usp14 in the increased peak cell density seen with miR-378-3p depletion. This study highlights miR-378-3p as a novel engineering candidate for improving CHO cell growth. The use of subcellular fractionation also improved proteome coverage in the identification of novel miRNA targets.

Background Docetaxel based therapy is one of the first line chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. However, one of the major obstacles in the treatment of these patients is docetaxel-resistance. Defining the mechanisms of resistance so as to inform subsequent treatment options and combinations represents a challenge for clinicians and scientists. Previous work by our group has shown complex changes in pro and anti-apoptotic proteins in the development of resistance to docetaxel. Targeting these changes individually does not significantly impact on the resistant phenotype but understanding the central signalling pathways and transcription factors (TFs) which control these could represent a more appropriate therapeutic targeting approach. Methods Using a number of docetaxel-resistant sublines of PC-3 cells, we have undertaken a transcriptomic analysis by expression microarray using the Affymetrix Human Gene 1.0 ST Array and in conjunction with bioinformatic analyses undertook to predict dysregulated TFs in docetaxel resistant prostate cancer. The clinical significance of this prediction was ascertained by performing immunohistochemical (IHC) analysis of an identified TF (SRF) in the metastatic sites from men who died of advanced CRPC. Investigation of the functional role of SRF was examined by manipulating SRF using SiRNA in a docetaxel-resistant PC-3 cell line model. Results The transcription factors identified include serum response factor (SRF), nuclear factor kappa-B (NFκB), heat shock factor protein 1 (HSF1), testicular receptor 2 & 4 (TR2 &4), vitamin-D and retinoid x receptor (VDR-RXR) and oestrogen-receptor 1 (ESR1), which are predicted to be responsible for the differential gene expression observed in docetaxel-resistance. IHC analysis to quantify nuclear expression of the identified TF SRF correlates with both survival from date of bone metastasis (p = 0.003), survival from androgen independence (p = 0.00002), and overall survival from prostate cancer (p = 0.0044). Functional knockdown of SRF by siRNA demonstrated a reversal of apoptotic resistance to docetaxel treatment in the docetaxel-resistant PC-3 cell line model. Conclusions Our results suggest that SRF could aid in treatment stratification of prostate cancer, and may also represent a therapeutic target in the treatment of men afflicted with advanced prostate cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3100-4) contains supplementary material, which is available to authorized users.

Recent sequencing of the Chinese hamster ovary (CHO) cell and Chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering. European Commission Horizon 2020 Science Foundation Ireland

Publication of Chinese hamster ovary (CHO) cell line and Chinese hamster genomes is accelerating efforts to increase the efficiency of biopharmaceutical manufacturing through greater understanding of CHO cell biology. It is hoped that this knowledge will lead to more predictable bioprocesses through the identification of biomarkers for culture monitoring and engineering of the CHO cell itself. If we are to translate the potential of the CHO systems biology era to industrial practice, the extraction of knowledge from complex genomic, proteomic, transcriptomic and metabolomic datasets will be critical. In this manuscript, we review the methods utilized to analyze expression profiling data and highlight the role of advanced statistics as we generate larger scale datasets and move toward integrated multi-omic analyses of the biological system.

Fundamental to the efficient production of quality biopharmaceuticals is the selection, optimization and tailored manipulation of the mammalian cellular production host. Engineering of these cell factories, predominantly the Chinese hamster ovary cell and advancements in bioprocess regimens have led to greatly increased product titres. The ability of miRNAs to regulate gene expression on a global level has generated considerable interest in these molecules as potential cell engineering targets. In this review, we briefly describe their organization and biogenesis and discuss their attributes as engineering tools in Chinese hamster ovary cells. The development of particular engineering strategies based upon further dissection of miRNA behavior will be considered, with particular emphasis on encouraging examples in Chinese hamster ovary cells and their potential for further development.