Portrait of Professor Martin Michaelis

Professor Martin Michaelis

Professor of Molecular Medicine
Biosciences Athena Swan Co-ordinator

About

Martin Michaelis received his Pharmacy Degree (Approbation, 1998) and his PhD (2001) from the Goethe-University, Frankfurt am Main, Germany. He then worked as postdoc and later deputy group leader in the research group of Professor Jindrich Cinatl at the Institute of Medical Virology (Goethe-University, Frankfurt am Main, Germany) and the Dr Petra Joh-Forschungshaus, a private research institute run by the Frankfurter Stiftung für krebskranke Kinder in Frankfurt am Main. In 2011, Martin joined the University of Kent. He runs a joint wet/ dry laboratory research group together with Dr Mark Wass.

ORCID ID: 0000-0002-5710-5888

Research interests

The research of Professor Michaelis is focused on the identification and investigation of drugs and their mechanisms of action. The primary interest lies on acquired drug resistance in cancer. In collaboration with Professor Jindrich Cinatl (Goethe-University, Frankfurt am Main), he manages and develops the Resistant Cancer Cell Line (RCCL) Collection a unique collection of >1,300 cancer cell lines with acquired resistance to anti-cancer drugs.
In addition, Professor Michaelis is interested in virulence mechanisms and therapeutic targets in viruses and in meta-research that investigates research practices in the life sciences.

Teaching

  • Virology - BI620  
  • Frontiers in Oncology - BI639
  • Cancer Biology - BI642 
  • The Molecular and Cellular Basis of Cancer - BI837 
  • Cancer Therapeutics - BI840 
  • Viral Pathogens - BI856 
  • Cancer Research in Focus - BI857 

Supervision

MSc-R project available for 2020/21

Investigating the determinants of SARS Coronavirus-2 pathogenicity (joint supervision with Dr Mark Wass)
Severe Acute Respiratory Coronavirus-2 (SARS-CoV-2) is currently causing a global pandemic with much of the world in a lockdown state to limit the spread of the virus and number of cases and deaths that it causes. There are now many thousands of SARS-CoV-2 genome sequences obtained from those infected. These can be analysed to advance our understanding of the genetic and molecular features that determine the properties of the virus. This project will focus on using computational approaches to compare the thousands of SARS-CoV-2 genome sequences with those of SARS-CoV, the related virus that caused the 2002-2003 SARS Coronavirus outbreak. While these two viruses are closely related there are important differences in the disease that they cause. For example, SARS-CoV-2 has a much lower death rate and appears to be more easily transmitted. We have already begun research in this area (see our preprint here: https://www.biorxiv.org/content/10.1101/2020.04.03.024257v1 
and this project will expand on this work. Additional research cost: £1500
Investigation of drug-adapted cancer cell lines joint supervision with Dr Mark Wass We host the Resistant Cancer Cell Line (RCCL) collection, the worldwide largest collection of drug-adapted cancer cell lines and models of acquired drug resistance in cancer at Kent. Here, drug-adapted cancer cell lines will be characterised and investigated to gain novel insights into the processes underlying resistance formation and to identify novel therapy candidates (including biomarkers). We offer wet lab and computational projects in this area. Additional research costs: £1500
Investigating determinants of virus pathogenicity joint supervision with Dr Mark Wass Our research has recently compared different species of Ebolaviruses to identify parts of their proteins that determine if they are pathogenic. This project will apply these computational approaches to different types of viruses (e.g. Zika virus, west Nile, human papillomavirus) to identify determinants of virus pathogenicity and gain insight into what make some viruses highly virulent while others are harmless. Additional research costs: £1500
Design of cancer cell-specific drug carrier systems (joint supervision Dr Christopher Serpell, School of Physical Sciences) The Serpell lab has produced perfectly sequence-defined polymers which self-assemble to give nanostructures with a remarkable variety of size and shape according to sequence and conditions (N. Appukutti, C. J. Serpell, Sequence Isomerism in Uniform Polyphosphoesters Programmes Self-Assembly and Folding, ChemRxiv, preprint posted 04.02.19, DOI: https://doi.org/10.26434/chemrxiv.7666316.v1. In this project, the effects of the polymer nanostructure on cell uptake and therapeutic efficacy will be studied in different cancer cells. This will provide pioneering insights into the prospects of sequence-defined polymers as carrier systems for anti-cancer drugs. Additional research costs: £1500 

Publications

Showing 75 of 177 total publications in the Kent Academic Repository. View all publications.

Forthcoming

  • Onafuye, H. (2020). Investigation of Drug-Adapted Cancer Cell Lines As Pre-Clinical Models of Acquired Resistance.
    Drug-adapted cancer cell lines have been successfully used to identify clinically relevant drug resistance mechanisms. This project focused on the further development of drug-adapted cancer cell lines as pre-clinical models of acquired drug resistance in cancer. A new cell line panel consisting of the ovarian cancer cell lines EFO-21, EFO-27, and COLO-704 and their cisplatin-adapted sublines was introduced and characterised. In addition, doxorubicin-loaded human serum albumin (HSA) nanoparticles were shown to circumvent ABCB1-mediated drug efflux. Vincristine- but not doxorubicin-adapted cells were re-sensitised to the level of the respective parental cells by HSA nanoparticle-incorporated doxorubicin. This indicates that rational strategies to overcome drug resistance in cancer depend on an intimate understanding of (the complexity of) the underlying resistance mechanisms. Finally, a standardised treatment protocol revealed differences in the potential of the microtubule-stabilising agents; docetaxel, paclitaxel, cabazitaxel, and epothilone B to induce resistance in the neuroblastoma cell line UKF-NB-3. In conclusion, this project has contributed to resistance research in cancer by introducing novel models, by providing novel insights into the prospects and limitations of strategies to overcome resistance mediated by transporter-mediated drug efflux, and by developing a novel strategy to assess the potential of anti-cancer drugs to induce resistance.

Article

  • McLaughlin, K., Bechtel, M., Bojkova, D., Münch, C., Ciesek, S., Wass, M., Michaelis, M. and Cinatl, J. (2020). COVID-19-Related Coagulopathy—Is Transferrin a Missing Link?. Diagnostics [Online] 10:539. Available at: https://doi.org/10.3390/diagnostics10080539.
    SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term “blood coagulation” in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term “blood coagulation”) was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations
  • Rothenburger, T., McLaughlin, K., Herold, T., Schneider, C., Oellerich, T., Rothweiler, F., Feber, A., Fenton, T., Wass, M., Keppler, O., Michaelis, M. and Cinatl, J. (2020). SAMHD1 is a key regulator of the lineage-specific response of acute lymphoblastic leukaemias to nelarabine. Communications Biology [Online] 3. Available at: https://doi.org/10.1038/s42003-020-1052-8.
    The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.
  • Michaelis, M., Wass, M., Reddin, I., Voges, Y., Rothweiler, F., Hehlgans, S., Cinatl, J., Mernberger, M., Nist, A., Stiewe, T., Rödel, F. and Cinatl Jr., J. (2020). YM155-Adapted Cancer Cell Lines Reveal Drug-Induced Heterogeneity and Enable the Identification of Biomarker Candidates for the Acquired Resistance Setting. Cancers [Online] 12. Available at: https://doi.org/10.3390/cancers12051080.
    Survivin is a drug target and its suppressant YM155 a drug candidate mainly investigated for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of 10 additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterized by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualized therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting.
  • Ward, J., Wu, Y., Harflett, C., Onafuye, H., Corol, D., Lomax, C., Macalpine, W., Cinatl Jr., J., Wass, M., Michaelis, M. and Beale, M. (2020). Miyabeacin: A new cyclodimer presents a potential role for willow in cancer therapy. Scientific Reports [Online] 10. Available at: https://doi.org/10.1038/s41598-020-63349-1.
    Willow (Salix spp.) is well known as a source of medicinal compounds, the most famous being salicin, the progenitor of aspirin. Here we describe the isolation, structure determination, and anti-cancer activity of a cyclodimeric salicinoid (miyabeacin) from S. miyabeana and S. dasyclados. We also show that the capability to produce such dimers is a heritable trait and how variation in structures of natural miyabeacin analogues is derived via cross-over Diels-Alder reactions from pools of ortho-quinol precursors. These transient ortho-quinols have a role in the, as yet uncharacterised, biosynthetic pathways around salicortin, the major salicinoid of many willow genotypes.
  • Michaelis, M., Voges, Y., Rothweiler, F., Weipert, F., Zia-Ahmad, A., Cinatl, J., von Deimling, A., Westermann, F., Rödel, F., Wass, M. and Cinatl, Jr., J. (2020). Testing of the Survivin Suppressant YM155 in a Large Panel of Drug-Resistant Neuroblastoma Cell Lines. Cancers [Online] 12:577. Available at: https://dx.doi.org/10.3390/cancers12030577.
    The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). Seventy seven (77) cell lines displayed YM155 IC\(_{50}\)s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, \(TP53\)-mutant cells were not generally less sensitive to YM155 than \(TP53\) wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.
  • Dilruba, S., Grondana, A., Schiedel, A., Ueno, N., Bartholomeusz, C., Cinatl Jr, J., McLaughlin, K., Wass, M., Michaelis, M. and Kalayda, G. (2020). Non-Phosphorylatable PEA-15 Sensitises SKOV-3 Ovarian Cancer Cells to Cisplatin. Cells [Online] 9:515. Available at: https://dx.doi.org/10.3390/cells9020515.
    The efficacy of cisplatin-based chemotherapy in ovarian cancer is often limited by the development of drug resistance. In most ovarian cancer cells, cisplatin activates extracellular signal-regulated kinase1/2 (ERK1/2) signalling. Phosphoprotein enriched in astrocytes (PEA-15) is a ubiquitously expressed protein, capable of sequestering ERK1/2 in the cytoplasm and inhibiting cell proliferation. This and other functions of PEA-15 are regulated by its phosphorylation status. In this study, the relevance of PEA-15 phosphorylation state for cisplatin sensitivity of ovarian carcinoma cells was examined. The results of MTT-assays indicated that overexpression of PEA-15AA (a non-phosphorylatable variant) sensitised SKOV-3 cells to cisplatin. Phosphomimetic PEA-15DD did not affect cell sensitivity to the drug. While PEA-15DD facilitates nuclear translocation of activated ERK1/2, PEA-15AA acts to sequester the kinase in the cytoplasm as shown by Western blot. Microarray data indicated deregulation of thirteen genes in PEA-15AA-transfected cells compared to non-transfected or PEA-15DD-transfected variants. Data derived from The Cancer Genome Atlas (TCGA) showed that the expression of seven of these genes including EGR1 (early growth response protein 1) and FLNA (filamin A) significantly correlated with the therapy outcome in cisplatin-treated cancer patients. Further analysis indicated the relevance of nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signalling for the favourable effect of PEA-15AA on cisplatin sensitivity. The results warrant further evaluation of the PEA-15 phosphorylation status as a potential candidate biomarker of response to cisplatin-based chemotherapy. View Full-Text
  • Vallo, S., Stege, H., Berg, M., Michaelis, M., Winkelmann, R., Rothweiler, F. and Cinatl, J. (2020). Tumor necrosis factor‑related apoptosis‑inducing ligand as a therapeutic option in urothelial cancer cells with acquired resistance against first‑line chemotherapy. Oncology Reports [Online] 43:1331-1337. Available at: https://doi.org/10.3892/or.2020.7487.
    Patients with urothelial carcinoma frequently fail to respond to first‑line chemotherapy using cisplatin and gemcitabine due to development of resistant tumor cells. The aim of the present study was to investigate whether an alternative treatment with tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) that induces tumor cell death via the extrinsic apoptotic pathway may be effective against chemotherapy‑resistant urothelial cancer cell lines. The viability of the urothelial cancer cell line RT112 and its chemotherapy‑adapted sublines was investigated by MTT assay. The expression of anti‑apoptotic proteins was determined by western blotting and the individual roles of cellular inhibitor of apoptosis protein (cIAP)1, cIAP2, x‑linked inhibitor of apoptosis protein (XIAP) and induced myeloid leukemia cell differentiation protein (Mcl‑1) were investigated by siRNA‑mediated depletion. In particular, the bladder cancer sublines that were resistant to gemcitabine and cisplatin were cross‑resistant to TRAIL. Resistant cells displayed upregulation of anti‑apoptotic molecules compared with the parental cell line. Treatment with the second mitochondrial activator of caspases (SMAC) mimetic LCL‑161 that antagonizes cIAP1, cIAP2 and XIAP resensitized chemoresistant cells to TRAIL. The resensitization of tumor cells to TRAIL was confirmed by depletion of antiapoptotic proteins with siRNA. Collectively, the findings of the present study demonstrated that SMAC mimetic LCL‑161 increased the sensitivity of the parental cell line RT112 and chemotherapy‑resistant sublines to TRAIL, suggesting that inhibiting anti‑apoptotic molecules renders TRAIL therapy highly effective for chemotherapy‑sensitive and ‑resistant urothelial cancer cells.
  • Vogel, J., Schmidt, S., Schmidt, D., Rothweiler, F., Koch, B., Baer, P., Rabenau, H., Michel, D., Stamminger, T., Michaelis, M. and Cinatl, J. (2019). The Thrombopoietin Receptor Agonist Eltrombopag Inhibits Human Cytomegalovirus Replication Via Iron Chelation. Cells [Online] 9:31-31. Available at: https://dx.doi.org/10.3390/cells9010031.
    The thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to the effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interfered with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and the addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after hematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses, and synergistically increase the effects of ganciclovir, eltrombopag is also a drug-repurposing candidate for the treatment of therapy-refractory HCMV disease.
  • Thomas, S., Balónová, B., Cinatl, J., Wass, M., Serpell, C., Blight, B. and Michaelis, M. (2019). Thiourea and Guanidine Compounds and their Iridium Complexes in Drug-Resistant Cancer Cell Lines: Structure-Activity Relationships and Direct Luminescent Imaging. ChemMedChem [Online]. Available at: https://doi.org/10.1002/cmdc.201900591.
    Thiourea and guanidine units are found in nature, medicine, and materials. Their continued exploration in applications as diverse as cancer therapy, sensors, and electronics means that their toxicity is an important consideration. We have systematically synthesised a set of thiourea compounds and their guanidine analogues, and elucidated structure-activity relationships in terms of cellular toxicity in three ovarian cancer cell lines and their cisplatin-resistant sub-lines. We have been able to use the intrinsic luminescence of iridium complexes to visualise the effect of both structure alteration and cellular resistance mechanisms. These findings provide starting points for the development of new drugs and consideration of safety issues for novel thiourea- and guanidine-based materials.
  • Michaelis, M., Kleinschmidt, M., Bojkova, D., Rabenau, H., Wass, M. and Cinatl Jr., J. (2019). Omeprazole Increases the Efficacy of Acyclovir Against Herpes Simplex Virus Type 1 and 2. Frontiers in Microbiology [Online] 10. Available at: https://dx.doi.org/10.3389/fmicb.2019.02790.
    Omeprazole was shown to improve the anti-cancer effects of the nucleoside analogue 5-fluorouracil. Here, we combined omeprazole with the antiviral nucleoside analogues ribavirin and acyclovir. Omeprazole did not affect the antiviral effects of ribavirin in non-toxic concentrations up to 80 μg/mL but increased the acyclovir-mediated effects on herpes simplex virus 1 and 2 (HSV-1 and -2) replication in a dose-dependent manner. Omeprazole alone reduced HSV-1 and -2 titers [but not HSV-induced formation of cytopathogenic effects (CPE)] at concentrations ≥40 μg/mL. However, it exerted substantially stronger effects on acyclovir activity and also increased acyclovir activity at lower concentrations that did not directly interfere with HSV replication. Omeprazole 80 μg/mL caused a 10.8-fold (Vero cells) and 47.7-fold (HaCaT cells) decrease of the acyclovir concentrations that reduced HSV-1-induced CPE formation by 50% (IC50). In HSV-2-infected cells, omeprazole 80 μg/mL reduced the acyclovir IC50 by 7.3- (Vero cells) and 12.9-fold (HaCaT cells). In HaCaT cells, omeprazole 80 μg/mL reduced the HSV-1 titer in the presence of acyclovir 1 μg/mL by 1.6 × 105-fold and the HSV-2 titer in the presence of acyclovir 2 μg/mL by 9.2 × 103-fold. The proton pump inhibitors pantoprazole, rabeprazole, lansoprazole, and dexlansoprazole increased the antiviral effects of acyclovir in a similar fashion as omeprazole, indicating this to be a drug class effect. In conclusion, proton pump inhibitors increase the anti-HSV activity of acyclovir and are candidates for antiviral therapies in combination with acyclovir, in particular for topical preparations for the treatment of immunocompromised individuals who are more likely to suffer from severe complications.
  • Pieper, S., Onafuye, H., Mulac, D., Cinatl Jr., J., Wass, M., Michaelis, M. and Langer, K. (2019). Incorporation of doxorubicin in different polymer nanoparticles and their anticancer activity. Beilstein Journal of Nanotechnology [Online] 10:2062-2072. Available at: https://dx.doi.org/10.3762/bjnano.10.201.
    Background: Nanoparticles are under investigation as carrier systems for anticancer drugs. The expression of efflux transporters such as the ATP-binding cassette (ABC) transporter ABCB1 is an important resistance mechanism in therapy-refractory cancer cells. Drug encapsulation into nanoparticles has been shown to bypass efflux-mediated drug resistance, but there are also conflicting results. To investigate whether easy-to-prepare nanoparticles made of well-tolerated polymers may circumvent transporter-mediated drug efflux, we prepared poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) nanoparticles loaded with the ABCB1 substrate doxorubicin by solvent displacement and emulsion diffusion approaches and assessed their anticancer efficiency in neuroblastoma cells, including ABCB1-expressing cell lines, in comparison to doxorubicin solution.

    Results: The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement led to the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH 7 reaching 53 µg doxorubicin/mg nanoparticle. These PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH 7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anticancer effects. However, nanoparticle-encapsulated doxorubicin did not display increased efficacy in ABCB1-expressing cells relative to doxorubicin solution.

    Conclusion: Doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anticancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anticancer efficacy. The design of drug-loaded nanoparticles with optimised anticancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anticancer therapy. Further research is required to understand under which circumstances nanoparticles can be used to overcome efflux-mediated resistance in cancer cells.
  • Povey, J., Saintas, E., Aderemi, A., Rothweiler, F., Zehner, R., Dirks, W., Cinatl, J., Racher, A., Wass, M., Smales, C. and Michaelis, M. (2019). Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines. Cells [Online] 8:1194. Available at: https://doi.org/10.3390/cells8101194.
    The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.
  • Onafuye, H., Pieper, S., Mulac, D., Jr., J., Wass, M., Langer, K. and Michaelis, M. (2019). Doxorubicin-Loaded Human Serum Albumin Nanoparticles Overcome Transporter-Mediated Drug Resistance in Drug-Adapted Cancer Cells. Beilstein Journal of Nanotechnology [Online] 10:1707-1715. Available at: https://doi.org/10.3762/bjnano.10.166.
    Resistance to systemic drug therapy is a major reason for the failure of anticancer therapies. Here, we tested doxorubicin-loaded human serum albumin (HSA) nanoparticles in the neuroblastoma cell line UKF-NB-3 and its ABCB1-expressing sublines adapted to vincristine (UKF-NB-3rVCR1) and doxorubicin (UKF-NB-3rDOX20). Doxorubicin-loaded nanoparticles displayed increased anticancer activity in UKF-NB-3rVCR1 and UKF-NB-3rDOX20 cells relative to doxorubicin solution, but not in UKF-NB-3 cells. UKF-NB-3rVCR1 cells were re-sensitised by nanoparticle-encapsulated doxorubicin to the level of UKF-NB-3 cells. UKF-NB-3rDOX20 cells displayed a more pronounced resistance phenotype than UKF-NB-3rVCR1 cells and were not re-sensitised by doxorubicin-loaded nanoparticles to the level of parental cells. ABCB1 inhibition using zosuquidar resulted in similar effects like nanoparticle incorporation, indicating that doxorubicin-loaded nanoparticles successfully circumvent ABCB1-mediated drug efflux. The limited re-sensitisation of UKF-NB-3rDOX20 cells to doxorubicin by circumvention of ABCB1-mediated efflux is probably due to the presence of multiple doxorubicin resistance mechanisms. So far, ABCB1 inhibitors have failed in clinical trials probably because systemic ABCB1 inhibition results in a modified body distribution of its many substrates including drugs, xenobiotics, and other molecules. HSA nanoparticles may provide an alternative, more specific way to overcome transporter-mediated resistance.
  • Oellerich, T., Schneider, C., Thomas, D., Knecht, K., Buzovetsky, O., Kaderali, L., Schliemann, C., Bohnenberger, H., Angenendt, L., Hartmann, W., Wardelmann, E., Rothenburger, T., Mohr, S., Scheich, S., Comoglio, F., Wilke, A., Ströbel, P., Serve, H., Michaelis, M., Ferreirós, N., Geisslinger, G., Xiong, Y., Keppler, O. and Cinatl, J. (2019). Selective inactivation of hypomethylating agents by SAMHD1 provides a rationale for therapeutic stratification in AML. Nature Communications [Online] 10. Available at: https://doi.org/10.1038/s41467-019-11413-4.
    Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.
  • Antczak, M., Michaelis, M. and Wass, M. (2019). Environmental conditions shape the nature of a minimal bacterial genome. Nature Communications [Online] 10. Available at: https://doi.org/10.1038/s41467-019-10837-2.
    Of the 473 genes in the genome of the bacterium with the smallest genome generated to date, 149 genes have unknown function, emphasising a universal problem; less than 1% of proteins have experimentally determined annotations. Here, we combine the results from state-of-the-art in silico methods for functional annotation and assign functions to 66 of the 149 proteins. Proteins that are still not annotated lack orthologues, lack protein domains, and/ or are membrane proteins. Twenty-four likely transporter proteins are identified indicating the importance of nutrient uptake into and waste disposal out of the minimal bacterial cell in a nutrient-rich environment after removal of metabolic enzymes. Hence, the environment shapes the nature of a minimal genome. Our findings also show that the combination of multiple different state-of-the-art in silico methods for annotating proteins is able to predict functions, even for difficult to characterise proteins and identify crucial gaps for further development.
  • Michaelis, M., Wass, M. and Cinatl, J. (2019). Drug-Adapted Cancer Cell Lines as Preclinical Models of Acquired Resistance. Cancer Drug Resistance [Online]. Available at: https://doi.org/10.20517/cdr.2019.005.
    Acquired resistance formation limits the efficacy of anti-cancer therapies. Acquired and intrinsic resistance differ conceptually. Acquired resistance is the consequence of directed evolution, whereas intrinsic resistance depends on the (stochastic) presence of pre-existing resistance mechanisms. Preclinical model systems are needed to study acquired drug resistance because they enable: (1) in depth functional studies; (2) the investigation of non-standard treatments for a certain disease condition (which is necessary to identify small groups of responders); and (3) the comparison of multiple therapies in the same system. Hence, they complement data derived from clinical trials and clinical specimens, including liquid biopsies. Many groups have successfully used drug-adapted cancer cell lines to identify and elucidate clinically relevant resistance mechanisms to targeted and cytotoxic anti-cancer drugs. Hence, we argue that drug-adapted cancer cell lines represent a preclinical model system in their own right that is complementary to other preclinical model systems and clinical data.
  • Martell, H., Masterson, S., McGreig, J., Michaelis, M. and Wass, M. (2019). Is the Bombali virus pathogenic in humans?. Bioinformatics [Online] 35:3553-3558. Available at: http://dx.doi.org/10.1093/bioinformatics/btz267.
    Motivation: The potential of the Bombali virus, a novel Ebolavirus, to cause disease in humans remains unknown. We have previously identified potential determinants of Ebolavirus pathogenicity in humans by analysing the amino acid positions that are differentially conserved (specificity 15 determining positions; SDPs) between human pathogenic Ebolaviruses and the non-pathogenic Reston virus. Here, we include the many Ebolavirus genome sequences that have since become available into our analysis and investigate the amino acid sequence of the Bombali virus proteins at the SDPs that discriminate between human pathogenic and non-human pathogenic Ebolaviruses. 20 Results: The use of 1408 Ebolavirus genomes (196 in the original analysis) resulted in a set of 166 SDPs (reduced from 180), 146 (88%) of which were retained from the original analysis. This indicates the robustness of our approach and refines the set of SDPs that distinguish human pathogenic Ebolaviruses from Reston virus. At SDPs, Bombali virus shared the majority of amino acids with the human pathogenic Ebolaviruses (63.25%). However, for two SDPs in VP24 (M136L, R139S) 25 that have been proposed to be critical for the lack of Reston virus human pathogenicity because they alter the VP24-karyopherin interaction, the Bombali virus amino acids match those of Reston virus. Thus, Bombali virus may not be pathogenic in humans. Supporting this, no Bombali virusassociated disease outbreaks have been reported, although Bombali virus was isolated from fruit bats cohabitating in close contact with humans, and anti-Ebolavirus antibodies that may indicate 30 contact with Bombali virus have been detected in humans.
  • Jossé, L., Bones, A., Purton, T., Michaelis, M. and Tsaousis, A. (2019). A Cell Culture Platform for the cultivation of Cryptosporidium parvum. Current Protocols in Microbiology [Online]. Available at: https://doi.org/10.1002/cpmc.80.
    Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhoea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the oesophageal cancer derived cell line COLO-680N, and can maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for four months. This chapter provides details on the methodologies used to culture, maintain and assess Cryptosporidium infection and propagation in COLO-680N.
  • Bones, A., Jossé, L., More, C., Miller, C., Michaelis, M. and Tsaousis, A. (2019). Past and future trends of Cryptosporidium in vitro research. Experimental Parasitology [Online] 196:28-37. Available at: 10.1016/j.exppara.2018.12.001.
    Cryptosporidium is a genus of single celled parasites capable of infecting a wide range of animals including humans. Cryptosporidium species are members of the phylum apicomplexa, which includes well-known genera such as Plasmodium and Toxoplasma. Cryptosporidium parasites cause a severe gastro-intestinal disease known as cryptosporidiosis. They are one of the most common causes of childhood diarrhoea worldwide, and infection can have prolonged detrimental effects on the development of children, but also can be life threatening to HIV/AIDS patients and transplant recipients. A variety of hosts can act as reservoirs, and Cryptosporidium can persist in the environment for prolonged times as oocysts. While there has been substantial interest in these parasites, there is very little progress in terms of treatment development and understanding the majority of the life cycle of this unusual organism. In this review, we will provide an overview on the existing knowledge of the biology of the parasite and the current progress in developing in vitro cultivation systems. We will then describe a synopsis of current and next generation approaches that could spearhead further research in combating the parasite.
  • Wass, M., Ray, L. and Michaelis, M. (2019). Understanding of researcher behaviour is required to improve data reliability. GigaScience [Online]. Available at: https://doi.org/10.1093/gigascience/giz017.
    Background
    A lack of data reproducibility (“reproducibility crisis”) has been extensively debated across many academic disciplines.

    Main body
    Although a reproducibility crisis is widely perceived, conclusive data on the scale of the problem and the underlying reasons are largely lacking. The debate is primarily focused on methodological issues. However, examples such as the use of misidentified cell lines illustrate that the availability of reliable methods does not guarantee good practice. Moreover, research is often characterised by a lack of established methods. Despite the crucial importance of researcher conduct, research and conclusive data on the determinants of researcher behaviour are widely missing.

    Conclusion
    Meta-research is urgently needed that establishes an understanding of the factors that determine researcher behaviour. This knowledge can then be used to implement and iteratively improve measures, which incentivise researchers to apply the highest standards resulting in high quality data.
  • Fenton, T., Garrett, M., Wass, M. and Michaelis, M. (2018). What really matters - response and resistance in cancer therapy. Cancer Drug Resistance [Online] 2018:200-203. Available at: https://doi.org/10.20517/cdr.2018.19.
  • Fenton, T., Garrett, M., Michaelis, M. and Wass, M. (2018). Meeting Abstracts of the BACR conference: response and resistance in cancer therapy. Cancer Drug Resistance [Online] 1:266-302. Available at: https://doi.org/10.20517/cdr.2018.18.
  • Jenks, A., Vyse, S., Wong, J., Kostaras, E., Keller, D., Burgoyne, T., Shoemark, A., Tsalikis, A., de la Roche, M., Michaelis, M., Cinatl, J., Huang, P. and Tanos, B. (2018). Primary Cilia Mediate Diverse Kinase Inhibitor Resistance Mechanisms in Cancer. Cell reports [Online] 23:3042-3055. Available at: http://dx.doi.org/10.1016/j.celrep.2018.05.016.
    Primary cilia are microtubule-based organelles that detect mechanical and chemical stimuli. Although cilia house a number of oncogenic molecules (including Smoothened, KRAS, EGFR, and PDGFR), their precise role in cancer remains unclear. We have interrogated the role of cilia in acquired and de novo resistance to a variety of kinase inhibitors, and found that, in several examples, resistant cells are distinctly characterized by an increase in the number and/or length of cilia with altered structural features. Changes in ciliation seem to be linked to differences in the molecular composition of cilia and result in enhanced Hedgehog pathway activation. Notably, manipulating cilia length via Kif7 knockdown is sufficient to confer drug resistance in drug-sensitive cells. Conversely, targeting of cilia length or integrity through genetic and pharmacological approaches overcomes kinase inhibitor resistance. Our work establishes a role for ciliogenesis and cilia length in promoting cancer drug resistance and has significant translational implications.
  • Masterson, S., Lobel, L., Carroll, M., Wass, M. and Michaelis, M. (2018). Herd Immunity to Ebolaviruses Is Not a Realistic Target for Current Vaccination Strategies. Frontiers in Immunology [Online] 9. Available at: https://doi.org/10.3389/fimmu.2018.01025.
    The recent West African Ebola virus pandemic, which affected >28,000 individuals increased interest in anti-Ebolavirus vaccination programs. Here, we systematically analyzed the requirements for a prophylactic vaccination program based on the basic reproductive number (R0, i.e. the number of secondary cases that result from an individual infection). Published R0 values were determined by systematic literature research and ranged from 0.37 to 20. R0s ?4 realistically reflected the critical early outbreak phases and superspreading events. Based on the R0, the herd immunity threshold (Ic) was calculated using the equation Ic=1–(1/R0). The critical vaccination coverage (Vc) needed to provide herd immunity was determined by including the vaccine effectiveness (E) using the equation Vc=Ic/E. At an R0 of 4, the Ic is 75% and at an E of 90%, more than 80% of a population need to be vaccinated to establish herd immunity. Such vaccination rates are currently unrealistic because of resistance against vaccinations, financial/ logistical challenges, and a lack of vaccines that provide long-term protection against all human-pathogenic Ebolaviruses. Hence, outbreak management will for the foreseeable future depend on surveillance and case isolation. Clinical vaccine candidates are only available for Ebola viruses. Their use will need to be focused on health care workers, potentially in combination with ring vaccination approaches.
  • Sarin, N., Engel, F., Rothweiler, F., Cinatl, J., Michaelis, M., Frötschl, R., Fröhlich, H. and Kalayda, G. (2018). Key Players of Cisplatin Resistance: Towards a Systems Pharmacology Approach. International Journal of Molecular Sciences [Online] 19:767-785. Available at: https://doi.org/10.3390/ijms19030767.
    The major obstacle in the clinical use of the antitumor drug cisplatin is inherent and
    acquired resistance. Typically, cisplatin resistance is not restricted to a single mechanism demanding
    for a systems pharmacology approach to understand a whole cell’s reaction to the drug. In this
    study, the cellular transcriptome of untreated and cisplatin-treated A549 non-small cell lung cancer
    cells and their cisplatin-resistant sub-line A549rCDDP2000 was screened with a whole genome array
    for relevant gene candidates. By combining statistical methods with available gene annotations
    and without a previously defined hypothesis HRas, MAPK14 (p38), CCL2, DOK1 and PTK2B
    were identified as genes possibly relevant for cisplatin resistance. These and related genes were
    further validated on transcriptome (qRT-PCR) and proteome (Western blot) level to select candidates
    contributing to resistance. HRas, p38, CCL2, DOK1, PTK2B and JNK3 were integrated into a model
    of resistance-associated signalling alterations describing differential gene and protein expression
    between cisplatin-sensitive and -resistant cells in reaction to cisplatin exposure.
  • Miller, C., Jossé, L., Brown, I., Povey, J., Yiangou, L., Price, M., Cinalt, J., Xue, W., Michaelis, M. and Tsaousis, A. (2018). A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology. International Journal for Parasitology [Online] 48:197-201. Available at: https://doi.org/10.1016/j.ijpara.2017.10.001.
    Cryptosporidium parasites are a major cause of diarrhoea that pose a particular threat to children in developing areas and immunocompromised individuals. Curative therapies and vaccines are lacking, mainly due to lack of a long-term culturing system of this parasite. Here, we show that COLO-680N cells infected with two different Cryptosporidium parvum strains produce sufficient infectious oocysts to infect subsequent cultures, showing a substantial fold increase in production, depending on the experiment, over the most optimistic HCT-8 models. Oocyst identity was confirmed using a variety of microscopic- and molecular-based methods. This culturing system will accelerate research on Cryptosporidium and the development of anti-Cryptosporidium drugs.
  • Das, C., Linder, B., Bonn, F., Rothweiler, F., Dikic, I., Michaelis, M., Cinatl, J., Mandal, M. and Kögel, D. (2018). BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells. Neoplasia [Online] 20:263-279. Available at: http://dx.doi.org/10.1016/j.neo.2018.01.001.
    Target-specific treatment modalities are currently not available for triple-negative breast cancer (TNBC), and acquired chemotherapy resistance is a primary obstacle for the treatment of these tumors. Here we employed derivatives of BT-549 and MDA-MB-468 TNBC cell lines that were adapted to grow in the presence of either 5-Fluorouracil, Doxorubicin or Docetaxel in an aim to identify molecular pathways involved in the adaptation to drug-induced cell killing. All six drug-adapted BT-549 and MDA-MB-468 cell lines displayed cross resistance to chemotherapy and decreased apoptosis sensitivity. Expression of the anti-apoptotic co-chaperone BAG3 was notably enhanced in two thirds (4/6) of the six resistant lines simultaneously with higher expression of HSP70 in comparison to parental controls. Doxorubicin-resistant BT-549 (BT-549DOX) and 5-Fluorouracil-resistant MDA-MB-468 (MDA-MB-4685-FU) cells were chosen for further analysis with the autophagy inhibitor Bafilomycin A1 and lentiviral depletion of ATG5, indicating that enhanced cytoprotective autophagy partially contributes to increased drug resistance and cell survival. Stable lentiviral BAG3 depletion was associated with a robust down-regulation of Mcl-1, Bcl-2 and Bcl-xL, restoration of drug-induced apoptosis and reduced cell adhesion in these cells, and these death-sensitizing effects could be mimicked with the BAG3/Hsp70 interaction inhibitor YM-1 and by KRIBB11, a selective transcriptional inhibitor of HSF-1. Furthermore, BAG3 depletion was able to revert the EMT-like transcriptional changes observed in BT-549DOX and MDA-MB-4685-FU cells. In summary, genetic and pharmacological interference with BAG3 is capable to resensitize TNBC cells to treatment, underscoring its relevance for cell death resistance and as a target to overcome therapy resistance of breast cancer.
  • Vallo, S., Rutz, J., Kautsch, M., Winkelmann, R., Michaelis, M., Wezel, F., Bartsch, G., Haferkamp, A., Rothweiler, F., Blaheta, R. and Cinatl, J. (2017). Blocking integrin ?1 decreases adhesion in chemoresistant urothelial cancer cell lines. Oncology letters [Online] 14:5513-5518. Available at: http://dx.doi.org/10.3892/ol.2017.6883.
    Treatment failure in metastatic bladder cancer is commonly caused by acquisition of resistance to chemotherapy in association with tumor progression. Since alterations of integrins can influence the adhesive and invasive behaviors of urothelial bladder cancer cell lines, the present study aimed to evaluate the role of integrins in bladder cancer cells with acquired resistance to standard first-line chemotherapy with gemcitabine, and cisplatin. Therefore, four gemcitabine- and four cisplatin-resistant sublines out of a panel of four parental urothelial bladder cancer cell lines (TCC-SUP, HT1376, T24, and 5637) were used. Expression of integrin subunits ?3, ?5, ?6, ?1, ?3, and ?4 was detected using flow cytometry. Adhesion and chemotaxis were analyzed. For functional assays, integrin ?1 was attenuated with a blocking antibody. In untreated cells, chemotaxis was upregulated in 3/4 gemcitabine-resistant sublines. In cisplatin-resistant cells, chemotaxis was enhanced in 2/4 cell lines. Acquired chemoresistance induced the upregulation of integrin ?1 in all four tested gemcitabine-resistant sublines, as well as an upregulation in 3/4 cisplatin-resistant sublines compared with parental cell lines. Following the inhibition of integrin ?1, adhesion to extracellular matrix components was downregulated in 3/4 gemcitabine-resistant sublines and in all four tested cisplatin-resistant sublines. Since integrin ?1 is frequently upregulated in chemoresistant urothelial cancer cell lines and inhibition of integrin ?1 may influence adhesion, further studies are warranted to evaluate integrin ?1 as a potential therapeutic target for bladder cancer.
  • Pappalardo, M., Collu, F., Macpherson, J., Michaelis, M., Fraternali, F. and Wass, M. (2017). Investigating Ebola virus pathogenicity using Molecular Dynamics. BMC Genomics [Online] 18. Available at: https://dx.doi.org/10.1186/s12864-017-3912-2.
    Background: Ebolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5.

    Results: As a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling.

    Conclusion: Our results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.
  • Ahmad, A., Sarin, N., Engel, F., Kalayda, G., Mannewitz, M., Cinatl, J., Rothweiler, F., Michaelis, M., Saafan, H., Ritter, C., Jaehde, U. and Frötschl, R. (2017). Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest. PLOS ONE [Online] 12:e0181081. Available at: http://dx.doi.org/10.1371/journal.pone.0181081.
    The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC) cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2), xeroderma pigmentosum complementation group C (XPC), stress inducible protein (SIP) and p21) compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm) and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis.
  • Pappalardo, M., Reddin, I., Cantoni, D., Rossman, J., Michaelis, M. and Wass, M. (2017). Changes associated with Ebola virus adaptation to novel species. Bioinformatics [Online] 33:1911-1915. Available at: http://dx.doi.org/10.1093/bioinformatics/btx065.
    Motivation: Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity.
    Results: We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24, and VP35. Only VP24, GP and NP were consistently found mutated in rodent-adapted Ebola virus strains. Fewer than five mutations in these genes seem to be required for the adaptation of Ebola viruses to a new species. The role of mutations in GP and NP is not clear. However, three VP24 mutations located in the protein interface with karyopherin 5 may enable VP24 to inhibit karyopherins and subsequently the host interferon response. Three further VP24 mutations change hydrogen bonding or cause conformational changes. Hence, there is evidence that few mutations including crucial mutations in VP24 enable Ebola virus adaptation to new hosts. Since Reston virus, the only non-human pathogenic Ebolavirus species circulates in pigs in Asia, this raises concerns that few mutations may result in novel human pathogenic Ebolaviruses.
  • Kollareddy, M., Sherrard, A., Park, J., Szemes, M., Gallacher, K., Melegh, Z., Oltean, S., Michaelis, M., Cinatl, J., Kaidi, A. and Malik, K. (2017). The small molecule inhibitor YK-4-279 disrupts mitotic progression of neuroblastoma cells, overcomes drug resistance and synergizes with inhibitors of mitosis. Cancer Letters [Online] 403:74-85. Available at: https://doi.org/10.1016/j.canlet.2017.05.027.
    Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a highrisk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the RasMEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers.
  • Vallo, S., K�ppR., Michaelis, M., Rothweiler, F., Bartsch, G., Brandt, M., Gust, K., Wezel, F., Blaheta, R., Haferkamp, A. and Cinatl, J. (2017). Resistance to nanoparticle albumin-bound paclitaxel is mediated by ABCB1 in urothelial cancer cells. Oncology Letters [Online] 13. Available at: https://doi.org/10.3892/ol.2017.5986.
    Nanoparticle albumin?bound (nab)-paclitaxel appears to exhibit better response rates in patients with metastatic urothelial cancer of the bladder whom are pretreated with nab-paclitaxel compared with conventional paclitaxel. Paclitaxel may induce multidrug resistance in patients with cancer, while the mechanisms of resistance against paclitaxel are manifold. These include reduced function of pro?apoptotic proteins, mutations of tubulin and overexpression of the drug transporter adenosine 5'?triphosphate?binding cassette transporter subfamily B, member 1 (ABCB1). To evaluate the role of ABCB1 in nab?paclitaxel resistance in urothelial cancer cells, the bladder cancer cell lines T24 and TCC?SUP, as well as sub?lines with acquired resistance against gemcitabine (T24rGEMCI20 and TCC?SUPrGEMCI20) and vinblastine (T24rVBL20 and TCC?SUPrVBL20) were examined. For the functional inhibition of ABCB1, multi-tyrosine kinase inhibitors with ABCB1?inhibiting properties, including cabozantinib and crizotinib, were used. Additional functional assessment was performed with cell lines stably transduced with a lentiviral vector encoding for ABCB1, and protein expression was determined by western blotting. It was indicated that cell lines overexpressing ABCB1 exhibited similar resistance profiles to nab?paclitaxel and paclitaxel. Cabozantinib and crizotinib sensitized tumor cells to nab?paclitaxel and paclitaxel in the same dose?dependent manner in cell lines overexpressing ABCB1, without altering the downstream signaling of tyrosine kinases. These results suggest that the overexpression of ABCB1 confers resistance to nab?paclitaxel in urothelial cancer cells. Additionally, small molecules may overcome resistance to anticancer drugs that are substrates of ABCB1.
  • Saintas, E., Abrahams, L., Ahmed, G., Ajakaiye, A., AlHumaidi, A., Ashmore-Harris, C., Clark, I., Dura, U., Fixmer, C., Ike-Morris, C., Mato Prado, M., Mccullough, D., Mishra, S., Schoeler, K., Timur, H., Williamson, M., Alatsatianos, M., Bahsoun, B., Blackburn, E., Hogwood, C., Lithgow, P., Rowe, M., Yiangou, L., Rothweiler, F., Cinatl jr, J., Zehner, R., Baines, A., Garrett, M., Gourlay, C., Griffin, D., Gullick, W., Hargreaves, E., Howard, M., Lloyd, D., Rossman, J., Smales, C., Tsaousis, A., von der Haar, T., Wass, M. and Michaelis, M. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE [Online] 12:e0172140. Available at: http://dx.doi.org/10.1371/journal.pone.0172140.
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • Isono, M., Hoffmann, M., Pinkerneil, M., Sato, A., Michaelis, M., Cinatl, J., Niegisch, G. and Schulz, W. (2017). Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine. Journal of Experimental & Clinical Cancer Research [Online] 36. Available at: http://dx.doi.org/10.1186/s13046-016-0473-1.
    Background: More effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer.

    Methods: Viability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by ?H2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs.

    Results: Combined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762.

    Conclusions: AZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.
  • Sarin, N., Engel, F., Kalayda, G., Frötschl, R., Cinatl, J., Rothweiler, F., Michaelis, M., Fröhlich, H. and Jaehde, U. (2017). Knowledge-based approach to identify key determinants of cisplatin sensitivity. International journal of clinical pharmacology and therapeutics [Online] 55:686-689. Available at: http://dx.doi.org/10.5414/CPXCES15EA04.
  • Popay, A., Lloyd, D., Wass, M. and Michaelis, M. (2017). Dexamethasone for the Prevention of Cisplatin-induced Ototoxicity. Clinical Cancer Drugs [Online] 4:59-64. Available at: https://doi.org/10.2174/2212697X04666170331171359.
    Background: Cisplatin is a commonly used anti-cancer drug. However, its use is associated with severe side effects including ototoxicity that affects a large fraction of cisplatin-treated patients. Approved therapies that reduce cisplatin-induced ototoxicity are lacking. Among the candidate therapeutics, dexamethasone stands out. There is extensive experience of its use in combination with cisplatin for the prevention of chemotherapy-induced nausea and vomiting indicating that dexamethasone does not affect the anti-cancer effects of cisplatin.

    Objective: The objective of this study is to assess the potential of dexamethasone for the prevention of cisplatin-induced ototoxicity by a systematic analysis of the available evidence.
    Method: The databases PubMed and Web of Science were used to identify relevant articles by using the search terms 'cisplatin', 'ototoxicity', and 'dexamethasone'.
    Results: We identified 16 relevant original research articles. The analyzed studies reported conflicting results on the effects of dexamethasone on cisplatin-induced ototoxicity. However, studies in which dexamethasone was used prior to cisplatin treatment and directly administered into the tympanic cavity of the middle ear consistently reported beneficial effects. The use of sustained release formulations that prolong the availability of dexamethasone within the ear further improved the efficacy of dexamethasone.
    Conclusion: Dexamethasone is a promising candidate drug for the prevention of cisplatin-induced ototoxicity when applied intratympanically. Optimized formulations and administration schedules with regard to dose and time of application need to be developed.
  • Cantoni, D., Hamlet, A., Michaelis, M., Wass, M. and Rossman, J. (2016). Risks Posed by Reston, the Forgotten Ebolavirus. mSphere [Online] 1. Available at: http://dx.doi.org/10.1128/mSphere.00322-16.
  • Schneider, C., Oellerich, T., Baldauf, H., Schwarz, S., Thomas, D., Flick, R., Bohnenberger, H., Kaderali, L., Stegmann, L., Cremer, A., Martin, M., Lohmeyer, J., Michaelis, M., Hornung, V., Schliemann, C., Berdel, W., Hartmann, W., Wardelmann, E., Comoglio, F., Hansmann, M., Yakunin, A., Geisslinger, G., Ströbel, P., Ferreirós, N., Serve, H., Keppler, O. and Cinatl, J. (2016). SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia. Nature medicine [Online] 23:250-255. Available at: http://dx.doi.org/10.1038/nm.4255.
    The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.
  • Shagari, H., Rossman, J., Wass, M. and Michaelis, M. (2016). The 2014 Ebola Outbreak: Preparedness in West African Countries and its Impact on the Size of the Outbreak. Journal of Emerging Diseases and Virology [Online] 2. Available at: http://dx.doi.org/10.16966/2473-1846.123.
    The recent Ebola virus outbreak in West Africa was the first that reached epidemic size resulting in more than 28,000 suspected and confirmed cases and more than 11,000 deaths. Here, we performed a meta-analysis to determine the role of preparedness in the course of the epidemic. Relevant research articles were identified using the search terms "Ebola 2014 preparedness", "Ebola 2014 treatment and diagnosis", "Ebola 2014 isolation", "Ebola 2014 culture", and "Ebola 2014 Health Care Workers" in PubMed. 21 relevant original articles in English were identified and analysed. Results revealed that a lack of preparedness substantially contributed to the scale of the Ebola epidemic in West Africa. Studies consistently reported on shortcomings in the availability and use of personal protective equipment, transportation and communication systems, surveillance, patient isolation and treatment, training of healthcare workers, and public awareness and perception in the affected West African countries. Effective surveillance and patient isolation enabled outbreak control. In conclusion, effective health care systems and procedures for early detection and containment of outbreaks, in combination with education of the population will be needed to better control future Ebola outbreaks and outbreaks of other (novel) pathogens for which no effective treatment is available.
  • Voges, Y., Michaelis, M., Rothweiler, F., Schaller, T., Schneider, C., Politt, K., Mernberger, M., Nist, A., Stiewe, T., Wass, M., Rödel, F. and Cinatl, J. (2016). Effects of YM155 on survivin levels and viability in neuroblastoma cells with acquired drug resistance. Cell death & disease [Online] 7:e2410. Available at: http://dx.doi.org/10.1038/cddis.2016.257.
    Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action.
  • Vallo, S., Michaelis, M., Gust, K., Black, P., Rothweiler, F., Kvasnicka, H., Blaheta, R., Brandt, M., Wezel, F., Haferkamp, A. and Cinatl, J. (2016). Dasatinib enhances tumor growth in gemcitabine-resistant orthotopic bladder cancer xenografts. BMC research notes [Online] 9:454. Available at: http://dx.doi.org/10.1186/s13104-016-2256-3.
    BACKGROUND

    Systemic chemotherapy with gemcitabine and cisplatin is standard of care for patients with metastatic urothelial bladder cancer. However, resistance formation is common after initial response. The protein Src is known as a proto-oncogene, which is overexpressed in various human cancers. Since there are controversial reports about the role of Src in bladder cancer, we evaluated the efficacy of the Src kinase inhibitor dasatinib in the urothelial bladder cancer cell line RT112 and its gemcitabine-resistant sub-line RT112(r)GEMCI(20) in vitro and in vivo.

    METHODS

    RT112 urothelial cancer cells were adapted to growth in the presence of 20 ng/ml gemcitabine (RT112(r)GEMCI(20)) by continuous cultivation at increasing drug concentrations. Cell viability was determined by MTT assay, cell growth kinetics were determined by cell count, protein levels were measured by western blot, and cell migration was evaluated by scratch assays. In vivo tumor growth was tested in a murine orthotopic xenograft model using bioluminescent imaging.

    RESULTS

    Dasatinib exerted similar effects on Src signaling in RT112 and RT112(r)GEMCI(20) cells but RT112(r)GEMCI(20) cells were less sensitive to dasatinib-induced anti-cancer effects (half maximal inhibitory concentration (IC50) of dasatinib in RT112 cells: 349.2 ± 67.2 nM; IC50 of dasatinib in RT112(r)GEMCI(20) cells: 1081.1 ± 239.2 nM). Dasatinib inhibited migration of chemo-naive and gemcitabine-resistant cells. Most strikingly, dasatinib treatment reduced RT112 tumor growth and muscle invasion in orthotopic xenografts, while it was associated with increased size and muscle-invasive growth in RT112(r)GEMCI(20) tumors.

    CONCLUSION

    Dasatinib should be considered with care for the treatment of urothelial cancer, in particular for therapy-refractory cases.
  • Williams, M., Sánchez, E., Aluri, E., Douglas, F., MacLaren, D., Collins, O., Cussen, E., Budge, J., Sanders, L., Michaelis, M., Smales, C., Cinatl, J., Lorrio, S., Krueger, D., de Rosales, R. and Corr, S. (2016). Microwave-assisted synthesis of highly crystalline, multifunctional iron oxide nanocomposites for imaging applications. RSC Advances [Online] 6:83520-83528. Available at: http://doi.org/10.1039/c6ra11819d.
    We report a reproducible single-step, microwave-assisted approach for the preparation of multifunctional magnetic nanocomposites comprising superparamagnetic iron oxide (Fe3O4) cores, a polyelectrolyte stabilizer and an organic dye with no requirement for post-processing. The stabilisers poly(sodium 4-styrenesulfonate) (PSSS) and sodium polyphosphate (SPP) have been thoroughly investigated and from analysis using electron microscopy, dynamic light scattering measurements, magnetic hysteresis and magnetic resonance (MR) imaging, we show that the higher degree of Fe3O4 nanoparticle crystallinity achieved with the PSSS stabiliser leads to enhanced magnetic behaviour and thus better contrast agent relaxivity compared to the less crystalline, poorly defined particles obtained when SPP is employed as a stabiliser. We also demonstrate the potential for obtaining a multifunctional magnetic-fluorescent nanocomposite using our microwave-assisted synthesis. In this manner, we demonstrate the intimate link between synthetic methodology (microwave heating with a polyelectrolyte stabilizer) and the resulting properties (particle size, shape, and magnetism) and how this underpins the functionality of the resulting nanocomposites as agents for biomedical imaging.
  • Michaelis, M., Rossman, J. and Wass, M. (2016). Computational analysis of Ebolavirus data: prospects, promises and challenges. Biochemical Society transactions [Online] 44:973-8. Available at: http://dx.doi.org/10.1042/BST20160074.
    The ongoing Ebola virus (also known as Zaire ebolavirus, a member of the Ebolavirus family) outbreak in West Africa has so far resulted in >28000 confirmed cases compared with previous Ebolavirus outbreaks that affected a maximum of a few hundred individuals. Hence, Ebolaviruses impose a much greater threat than we may have expected (or hoped). An improved understanding of the virus biology is essential to develop therapeutic and preventive measures and to be better prepared for future outbreaks by members of the Ebolavirus family. Computational investigations can complement wet laboratory research for biosafety level 4 pathogens such as Ebolaviruses for which the wet experimental capacities are limited due to a small number of appropriate containment laboratories. During the current West Africa outbreak, sequence data from many Ebola virus genomes became available providing a rich resource for computational analysis. Here, we consider the studies that have already reported on the computational analysis of these data. A range of properties have been investigated including Ebolavirus evolution and pathogenicity, prediction of micro RNAs and identification of Ebolavirus specific signatures. However, the accuracy of the results remains to be confirmed by wet laboratory experiments. Therefore, communication and exchange between computational and wet laboratory researchers is necessary to make maximum use of computational analyses and to iteratively improve these approaches.
  • Löschmann, N., Michaelis, M., Rothweiler, F., Voges, Y., Balónová, B., Blight, B. and Cinatl, J. (2016). ABCB1 as predominant resistance mechanism in cells with acquired SNS-032 resistance. Oncotarget [Online] 7:58051-58064. Available at: http://dx.doi.org/10.18632/oncotarget.11160.
    The CDK inhibitor SNS-032 had previously exerted promising anti-neuroblastoma activity via CDK7 and 9 inhibition. ABCB1 expression was identified as major determinant of SNS-032 resistance. Here, we investigated the role of ABCB1 in acquired SNS-032 resistance. In contrast to ABCB1-expressing UKF-NB-3 sub-lines resistant to other ABCB1 substrates, SNS-032-adapted UKF-NB-3 (UKF-NB-3rSNS- 032300nM) cells remained sensitive to the non-ABCB1 substrate cisplatin and were completely re-sensitized to cytotoxic ABCB1 substrates by ABCB1 inhibition. Moreover, UKF-NB-3rSNS-032300nM cells remained similarly sensitive to CDK7 and 9 inhibition as UKF-NB-3 cells. In contrast, SHEPrSNS-0322000nM, the SNS-032-resistant sub-line of the neuroblastoma cell line SHEP, displayed low level SNS-032 resistance also when ABCB1 was inhibited. This discrepancy may be explained by the higher SNS-032 concentrations that were used to establish SHEPrSNS-0322000nM cells, since SHEP cells intrinsically express ABCB1 and are less sensitive to SNS-032 (IC50 912 nM) than UKF-NB-3 cells (IC50 153 nM). In conclusion, we show that ABCB1 expression represents the primary (sometimes exclusive) resistance mechanism in neuroblastoma cells with acquired resistance to SNS-032. Thus, ABCB1 inhibitors may increase the SNS-032 efficacy in ABCB1-expressing cells and prolong or avoid resistance formation.
  • Saafan, H., Foerster, S., Parra-Guillen, Z., Hammer, E., Michaelis, M., Cinatl, J., Völker, U., Fröhlich, H., Kloft, C. and Ritter, C. (2016). Utilising the EGFR interactome to identify mechanisms of drug resistance in non-small cell lung cancer - Proof of concept towards a systems pharmacology approach. European journal of pharmaceutical sciences [Online] 94:20-32. Available at: http://dx.doi.org/10.1016/j.ejps.2016.04.025.
    Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006(r)ERLO(0.5) adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006(r)ERLO(0.5) cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006(r)ERLO(0.5) cells. Taken together, differential proteins in the EGFR interactome of HCC4006(r)ERLO(0.5) cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance.
  • Wass, M., Rossman, J. and Michaelis, M. (2016). Ebola outbreak highlights the need for wet and dry laboratory collaboration. Journal of Virology and Emerging Diseases [Online] 2. Available at: http://dx.doi.org/10.16966/2473-1846.e102.
  • Wass, M., Rossman, J. and Michaelis, M. (2016). Ebola outbreak highlights the need for wet and dry laboratory collaboration. Journal of Emerging Diseases and Virology [Online] 2. Available at: http://dx.doi.org/10.16966/2473-1846.e102.
    The recent Ebola outbreak in Western Africa taught us that Ebolaviruses
    can cause much larger outbreaks and represent a much greater health
    threat than many of us believed (or wanted to believe). As of 30th March,
    the outbreak had resulted in 28,646 confirmed cases and 11,323 deaths.
    Although the WHO stated that the Ebola epidemic in West Africa no
    longer represents a Public Health Emergency of International Concern,
    since Guinea, Liberia, and Sierra are now capable of controlling and
    maintaining further small outbreaks, flare-ups still occur, most recently,
    on 4th April when two new cases were reported in Liberia (www.who.int).
  • Pappalardo, M., Juliá, M., Howard, M., Rossman, J., Michaelis, M. and Wass, M. (2016). Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses. Scientific reports [Online] 6:23743. Available at: http://dx.doi.org/10.1038/srep23743.
    Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission.
  • Michaelis, M., Rothweiler, F., Wurglics, M., Aniceto, N., Dittrich, M., Zettl, H., Wiese, M., Wass, M., Ghafourian, T., Schubert-Zsilavecz, M. and Cinatl, J. (2016). Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport. Oncotarget [Online] 7:11664-76. Available at: http://dx.doi.org/10.18632/oncotarget.7345.
    Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPAR?, PPAR?, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPAR?, and PPAR? represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPAR?, or PPAR?. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2?M with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport.
  • Dilruba, S., Michaelis, M., Cinatl, J. and Kalayda, G. (2015). Relevance of subcellular localization of extracellular signal-regulated kinase 1/2 (ERK1/2) for cisplatin resistance. International journal of clinical pharmacology and therapeutics [Online] 53:1041-5. Available at: http://www.dx.doi.org/10.5414/CPXCES14EA03.
  • Martin, M. and Cinatl, J. (2015). Cytomegalovirus and Cancer: A Long and Winding Road to Conclusive Evidence. Journal of Virology and Emerging Diseases [Online] 1:1-3. Available at: http://www.dx.doi.org/10.16966/jved.106.
  • Michaelis, M., Rothweiler, F., Löschmann, N., Sharifi, M., Ghafourian, T. and Cinatl, J. (2015). Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status. Oncotarget [Online] 6:17605-20. Available at: http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path[]=2889&pubmed-linkout=1.
    The PKC? inhibitor enzastaurin was tested in parental neuroblastoma and rhabdomyosarcoma cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53-depleted cells. Enzastaurin IC50s ranged from 3.3 to 9.5 ?M in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125 ?M interfered with ABCB1-mediated drug transport. PKC? and PKC? may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations ? 1.25 ?M. The investigated cell lines did not express PKC?. PKC? depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. These findings are important for the further development of therapies combining enzastaurin with ABC transporter substrates.
  • Michaelis, M., Mittelbronn, M. and Cinatl, J. (2015). Towards an unbiased, collaborative effort to reach evidence about the presence of human cytomegalovirus in glioblastoma (and other tumors). Neuro-oncology [Online] 17:1039. Available at: http://dx.doi.org/10.1093/neuonc/nov048.
  • Vallo, S., Michaelis, M., Rothweiler, F., Bartsch, G., Gust, K., Limbart, D., Rödel, F., Wezel, F., Haferkamp, A. and Cinatl, J. (2015). Drug-Resistant Urothelial Cancer Cell Lines Display Diverse Sensitivity Profiles to Potential Second-Line Therapeutics. Translational oncology [Online] 8:210-216. Available at: http://dx.doi.org/10.1016/j.tranon.2015.04.002.
    Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUP(r)GEMCI(20)), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer.
  • Mani, J., Vallo, S., Rakel, S., Antonietti, P., Gessler, F., Blaheta, R., Bartsch, G., Michaelis, M., Cinatl, J., Haferkamp, A. and Kögel, D. (2015). Chemoresistance is associated with increased cytoprotective autophagy and diminished apoptosis in bladder cancer cells treated with the BH3 mimetic (-)-Gossypol (AT-101). BMC cancer [Online] 15:224. Available at: http://dx.doi.org/10.1186/s12885-015-1239-4.
    BACKGROUND

    Acquired resistance to standard chemotherapy causes treatment failure in patients with metastatic bladder cancer. Overexpression of pro-survival Bcl-2 family proteins has been associated with a poor chemotherapeutic response, suggesting that Bcl-2-targeted therapy may be a feasible strategy in patients with these tumors. The small-molecule pan-Bcl-2 inhibitor (-)-gossypol (AT-101) is known to induce apoptotic cell death, but can also induce autophagy through release of the pro-autophagic BH3 only protein Beclin-1 from Bcl-2. The potential therapeutic effects of (-)-gossypol in chemoresistant bladder cancer and the role of autophagy in this context are hitherto unknown.

    METHODS

    Cisplatin (5637(r)CDDP(1000), RT4(r)CDDP(1000)) and gemcitabine (5637(r)GEMCI(20), RT4(r)GEMCI(20)) chemoresistant sub-lines of the chemo-sensitive bladder cancer cell lines 5637 and RT4 were established for the investigation of acquired resistance mechanisms. Cell lines carrying a stable lentiviral knockdown of the core autophagy regulator ATG5 were created from chemosensitive 5637 and chemoresistant 5637(r)GEMCI(20) and 5637(r)CDDP(1000) cell lines. Cell death and autophagy were quantified by FACS analysis of propidium iodide, Annexin and Lysotracker staining, as well as LC3 translocation.

    RESULTS

    Here we demonstrate that (-)-gossypol induces an apoptotic type of cell death in 5637 and RT4 cells which is partially inhibited by the pan-caspase inhibitor z-VAD. Cisplatin- and gemcitabine-resistant bladder cancer cells exhibit enhanced basal and drug-induced autophagosome formation and lysosomal activity which is accompanied by an attenuated apoptotic cell death after treatment with both (-)-gossypol and ABT-737, a Bcl-2 inhibitor which spares Mcl-1, in comparison to parental cells. Knockdown of ATG5 and inhibition of autophagy by 3-MA had no discernible effect on apoptotic cell death induced by (-)-gossypol and ABT-737 in parental 5637 cells, but evoked a significant increase in early apoptosis and overall cell death in BH3 mimetic-treated 5637(r)GEMCI(20) and 5637(r)CDDP(1000) cells.

    CONCLUSIONS

    Our findings show for the first time that (-)-gossypol concomitantly triggers apoptosis and a cytoprotective type of autophagy in bladder cancer and support the notion that enhanced autophagy may underlie the chemoresistant phenotype of these tumors. Simultaneous targeting of Bcl-2 proteins and the autophagy pathway may be an efficient new strategy to overcome their "autophagy addiction" and acquired resistance to current therapy.
  • Michaelis, M., Agha, B., Rothweiler, F., Löschmann, N., Voges, Y., Mittelbronn, M., Starzetz, T., Harter, P., Abhari, B., Fulda, S., Westermann, F., Riecken, K., Spek, S., Langer, K., Wiese, M., Dirks, W., Zehner, R., Cinatl, J., Wass, M. and Cinatl, J. (2015). Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen. Scientific reports [Online] 5:8202. Available at: http://www.nature.com/srep/2015/150203/srep08202/full/srep08202.html.
    Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.
  • Michaelis, M., Selt, F., Rothweiler, F., Wiese, M. and Cinatl, J. (2015). ABCG2 impairs the activity of the aurora kinase inhibitor tozasertib but not of alisertib. BMC research notes [Online] 8:484. Available at: http://www.biomedcentral.com/1756-0500/8/484.
    BACKGROUND

    Recently, we have shown that the ATP-binding cassette (ABC) transporter ABCB1 interferes with the anti-cancer activity of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) but not of the aurora kinase A and B inhibitor alisertib (MLN8237). Preliminary data had suggested tozasertib also to be a substrate of the ABC transporter ABCG2, another ABC transporter potentially involved in cancer cell drug resistance. Here, we studied the effect of ABCG2 on the activity of tozasertib and alisertib.

    RESULTS

    The tozasertib concentration that reduces cell viability by 50 % (IC50) was dramatically increased in ABCG2-transduced UKF-NB-3(ABCG2) cells (48.8-fold) compared to UKF-NB-3 cells and vector-transduced control cells. The ABCG2 inhibitor WK-X-34 reduced tozasertib IC50 to the level of non-ABCG2-expressing UKF-NB-3 cells. Furthermore, ABCG2 depletion from UKF-NB-3(ABCG2) cells using another lentiviral vector expressing an shRNA against the bicistronic mRNA of ABCG2 and eGFP largely re-sensitised these cells to tozasertib. In contrast, alisertib activity was not affected by ABCG2 expression.

    CONCLUSIONS

    Tozasertib but not alisertib activity is affected by ABCG2 expression. This should be considered within the design and analysis of experiments and clinical trials investigating these compounds.
  • Tunjung, W., Cinatl, J., Michaelis, M. and Smales, C. (2015). Anti-Cancer Effect of Kaffir Lime (Citrus Hystrix DC) Leaf Extract in Cervical Cancer and Neuroblastoma Cell Lines. Procedia Chemistry [Online] 14:465-468. Available at: http://doi.org/10.1016/j.proche.2015.03.062.
    Previous study showed that kaffir lime leaf contains alkaloid, flavonoid, terpenoid, tannin and saponin. The objective of this study was to examine the cytotoxic effect of kaffir lime leaf extract on cervical cancer and neuroblastoma cell lines. The method used for this research to determine cell viability was an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results showed that an ethyl acetate extract had an IC50 for HeLa cells, UKF-NB3, IMR-5 and SK-N-AS parental cells of 40.7 ?g · mL–1, 28.4 ?g · mL–1, 14.1 ?g · mL–1, and 25.2 ?g · mL–1 respectively. Furthermore, the IC50 of chloroform extracts for HeLa cells, UKF-NB3, IMR-5 and SK-N-AS parental were 17.6 ?g · mL–1, 18.9 ?g · mL–1, 6.4 ?g · mL–1, and 9.4 ?g · mL–1 respectively. These data showed that kaffir lime extract reduces the viability of cervical and neuroblastoma cell lines and may have potential as anti-cancer compounds.
  • Baumgarten, P., Michaelis, M., Rothweiler, F., Starzetz, T., Rabenau, H., Berger, A., Jennewein, L., Braczynski, A., Franz, K., Seifert, V., Steinbach, J., Allwinn, R., Mittelbronn, M. and Cinatl, J. (2014). Human cytomegalovirus infection in tumor cells of the nervous system is not detectable with standardized pathologico-virological diagnostics. Neuro-oncology [Online] 16:1469-77. Available at: http://neuro-oncology.oxfordjournals.org/content/16/11/1469.long.
    BACKGROUND

    Experimental findings have suggested that human cytomegalovirus (HCMV) infection of tumor cells may exert oncomodulatory effects that enhance tumor malignancy. However, controversial findings have been published on the presence of HCMV in malignant tumors. Here, we present the first study that systematically investigates HCMV infection in human nervous system tumors by highly sensitive immunohistochemistry in correlation with the HCMV serostatus of the patients.

    METHODS

    Immunohistochemical and quantitative PCR-based methods to detect different HCMV antigens and genomic HCMV DNA were optimized prior to the investigation of pathological samples. Moreover, the pathological results were matched with the HCMV serostatus of the patients.

    RESULTS

    HCMV immediate-early, late, and pp65 antigens could be detected in single cells from HCMV strain Hi91-infected UKF-NB-4 neuroblastoma cells after 1:1024 dilution with noninfected UKF-NB-4 cells. Genomic HCMV DNA could be detected in copy numbers as low as 430 copies/mL. However, we did not detect HCMV in tumors from a cohort of 123 glioblastoma, medulloblastoma, or neuroblastoma patients. Notably, we detected nonspecifically positive staining in tumor tissues of HCMV seropositive and seronegative glioblastoma patients. The HCMV serostatus of 67 glioblastoma patients matched the general epidemiological prevalence data for Western countries (72% of female and 57% of male glioblastoma patients were HCMV seropositive). Median survival was not significantly different in HCMV seropositive versus seronegative glioblastoma patients.

    CONCLUSIONS

    The prevalence of HCMV-infected tumor cells may be much lower than previously reported based on highly sensitive detection methods.
  • Michaelis, M., Rothweiler, F., Nerreter, T., van Rikxoort, M., Zehner, R., Dirks, W., Wiese, M. and Cinatl, J. (2014). Association between acquired resistance to PLX4032 (vemurafenib) and ATP-binding cassette transporter expression. BMC research notes [Online] 7:710-710. Available at: http://dx.doi.org/10.1186/1756-0500-7-710.
    BACKGROUND

    Various kinase inhibitors are known to be ATP-binding cassette (ABC) transporter substrates and resistance acquisition to kinase inhibitors has been associated to increased ABC transporter expression. Here, we investigated the role of the ABC transporters ABCB1, ABCC1, and ABCG2 during melanoma cell resistance acquisition to the V600-mutant BRAF inhibitors PLX4032 (vemurafenib) and PLX4720. PLX4032 had previously been shown to interfere with ABCB1 and ABCG2. PLX4720 had been demonstrated to interact with ABCB1 but to a lower extent than PLX4032.

    FINDINGS

    PLX4032 and PLX4720 affected ABCC1- and ABCG2-mediated drug transport in a similar fashion. In a panel of 16 V600E BRAF-mutated melanoma cell lines consisting of four parental cell lines and their sub-lines with acquired resistance to PLX4032, PLX4720, vincristine (cytotoxic ABCB1 and ABCC1 substrate), or mitoxantrone (cytotoxic ABCG2 substrate), we detected enhanced ABC transporter expression in 4/4 cytotoxic ABC transporter substrate-resistant, 3/4 PLX4720-resistant, and 1/4 PLX4032-resistant melanoma cell lines.

    CONCLUSION

    PLX4032 has the potential to induce ABC transporter expression but this potential is lower than that of PLX4720 or cytotoxic ABC transporter substrates. Since ABC transporters confer multi-drug resistance, this is of relevance for the design of next-line therapies.
  • Michaelis, M., Selt, F., Rothweiler, F., Löschmann, N., Nüsse, B., Dirks, W., Zehner, R. and Cinatl, J. (2014). Aurora kinases as targets in drug-resistant neuroblastoma cells. PloS one [Online] 9. Available at: http://dx.doi.org/10.1371/journal.pone.0108758.
    Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.
  • Cinatl, J., Speidel, D., Hardcastle, I. and Michaelis, M. (2014). Resistance acquisition to MDM2 inhibitors. Biochemical Society transactions [Online] 42:752-757. Available at: http://dx.doi.org/10.1042/BST20140035.
    Various experimental strategies aim to (re)activate p53 signalling in cancer cells. The most advanced clinically are small-molecule inhibitors of the autoregulatory interaction between p53 and MDM2 (murine double minute 2). Different MDM2 inhibitors are currently under investigation in clinical trials. As for other targeted anti-cancer therapy approaches, relatively rapid resistance acquisition may limit the clinical efficacy of MDM2 inhibitors. In particular, MDM2 inhibitors were shown to induce p53 mutations in experimental systems. In the present article, we summarize what is known about MDM2 inhibitors as anti-cancer drugs with a focus on the acquisition of resistance to these compounds.
  • Michaelis, M., Sithisarn, P. and Cinatl, J. (2014). Effects of flavonoid-induced oxidative stress on anti-H5N1 influenza a virus activity exerted by baicalein and biochanin A. BMC research notes [Online] 7:384-384. Available at: http://dx.doi.org/10.1186/1756-0500-7-384.
    BACKGROUND

    Different flavonoids are known to interfere with influenza A virus replication. Recently, we showed that the structurally similar flavonoids baicalein and biochanin A inhibit highly pathogenic avian H5N1 influenza A virus replication by different mechanisms in A549 lung cells. Here, we investigated the effects of both compounds on H5N1-induced reactive oxygen species (ROS) formation and the role of ROS formation during H5N1 replication.

    FINDINGS

    Baicalein and biochanin A enhanced H5N1-induced ROS formation in A549 cells and primary human monocyte-derived macrophages. Suppression of ROS formation induced by baicalein and biochanin A using the antioxidant N-acetyl-L-cysteine strongly increased the anti-H5N1 activity of both compounds in A549 cells but not in macrophages.

    CONCLUSIONS

    These findings emphasise that flavonoids induce complex pharmacological actions some of which may interfere with H5N1 replication while others may support H5N1 replication. A more detailed understanding of these actions and the underlying structure-activity relationships is needed to design agents with optimised anti-H5N1 activity.

Thesis

  • Smith, N. (2017). Investigating the Role of H2A.X Di-Phosphorylation in the DNA Damage Response.
    One of the earliest responses to DNA double strand breaks is phosphorylation of the C-terminal tail of histone variant H2A.X at position S139. The phosphorylated form (?H2A.X) is a marker of DNA damage and promotes DNA damage repair (DDR). Recent work showed that H2A.X can also be phosphorylated at position Y142, and that increasing Y142 phosphorylation by siRNA knockdown of the Eyes Absent (Eya) phosphotyrosine phosphatases (which remove Y142 phosphates) leads to increased apoptosis in response to DNA damage. This led to the proposal that an epigenetic "switch" between mono-?H2A.X (S139 only phosphorylated) and di-?H2A.X (S139 and Y142 both phosphorylated) directs the choice between DNA repair vs apoptosis in response to DNA damage. In this study, we sought to test this "switch" hypothesis by testing for cooperativity between the DNA damaging drug, Bleomycin, and Benzbromarone - a pharmacological inhibitor of Eya phosphatases. Although little cooperativity between Bleomycin and Benzbromarone was found in cell proliferation assays, live-cell imaging with fluorescent markers of apoptosis showed an increase in cell death. Moreover, by using novel antibodies that specifically recognise the various phosphorylated forms of H2A.X, immunochemical staining revealed that di-?H2A.X is located adjacent to and "flanks" ?H2A.X in DDR foci, and is interspersed with ?H2A.X in apoptotic cells. Therefore, contrary to the "switch" hypothesis being the singular function of di-?H2A.X, these results suggest an additional model for the role of di-?H2A.X in DDR, in that it may also act as a limiting factor in the rate of expansion of ?H2A.X repair foci.
  • Toutoudaki, A. (2017). Investigating Genetic Mechanisms of Cisplatin Resistance in Neuroblastoma Cell Lines.
    Cancer therapy development has improved the survival rate of patients over the past few decades. The major limitation of this approach is the inevitable emergence of resistance upon patient relapse. Acquired drug resistance can develop over the course of treatment causing tumours to no longer respond to therapy. There are several mechanisms that have been proposed to contribute to this development. Platinum compounds, a major class of chemotherapy agents, are used in approximately half of all chemotherapy schedules. Cisplatin is an alkylating-like agent that causes DNA damage by binding to purine bases and forming DNA lesions, eventually leading to cell death. Regardless of cisplatin's efficacy, there two major limitations for its use, severe cytotoxic side effects and the inevitable formation of drug resistance. For this project, two neuroblastoma cell lines UKF-NB-3 and UKF-NB-6, derived from late-stage high-risk neuroblastoma patients, have been adapted to cisplatin in order to investigate the emergence of resistance. Using next generation sequencing analysis tools, the parental sensitive cell lines have been analysed and compared to their cisplatin resistant sublines in order to identify key variations that could account for the emergence of resistance to cisplatin. In both resistant sublines, mutations in solute carrier proteins and calcium gated channels were found to be increased, suggesting effects on drug uptake and adaptation to tumour microenvironment. In addition, only in UKF-NB-3rCDDP1000, pathways associated with the structure and synthesis of the extracellular matrix where highly mutated which is predicted to alter drug uptake and efflux. Finally, in UKF-NB-6rCDDP2000 affected pathways included surface MHC-1 downregulation and dysregulation of glycogen metabolism, both predicted to contribute to the emergence of cisplatin resistance.
  • Jackson-Soutter, T. (2017). The Study of Acquired Drug Resistance in Cancer Cell Lines.
    Cytotoxic drugs are frequently used in the treatment of cancer. However whilst most patients initially respond well to cytotoxic therapies, acquired resistance often develops. In this project we aim to improve understanding of the mechanisms of acquired drug resistance and contribute to the development of a novel assay for determining the potential of different cancer drugs to cause acquired resistance.
    UKF-NB-3 cell lines, initially sensitive to docetaxel, paclitaxel, cabazitaxel and epothilone-b, were cultivated in the presence of each drug. Once resistance was conferred in the surviving sub-lines, the concentration of drug was increased to determine the extent of resistance. The sub-lines of UKF-NB-3 demonstrated resistance to Docetaxel and Paclitaxel. However, cabazitaxel and epothilone-b sub-lines were unable to adapt to the respective drugs. This demonstrates that resistance is more easily acquired to docetaxel and paclitaxel than cabazitaxel and epothilone b. To investigate the resistance mechanism responsible, cross resistance against a panel of selected cytotoxic drugs was determined. The surviving cell lines were resistant to additional drugs with high affinity to ABCB1 transporters. The expression of the drug efflux proteins (ABCB1) and tubulin was determined using confocal microscopy and found to be overexpressed in the resistant sub-lines.
    This project will lead to a standard protocol for determining the potential of anti-cancer drugs to cause acquired resistance in cancer cells. In this study we have acquired evidence that cultivation of UKF-NB-3 cell line in higher concentrations of docetaxel can result in increased degrees of resistance, possibly depending on the heterogeneity of the cell population. We have additionally characterised the mechanism of resistance through investigation of ATP-binding cassette (ABC) transporters and tubulin expression. With further work, this project will lead to improved understanding of resistance to tubulin binding agents, the cellular pathways responsible and ultimately improved therapeutic strategies and for drug adaptation of cell lines.
  • Bones, A. (2017). Developing in Vitro Culturing Techniques of the Apicomplexan Parasite Cryptosporidium Parvum.
    Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa, whose members are parasites of the GI tract and airways. Cryptosporidium is the second largest cause of childhood diarrhoea in children under two and is associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods, in particular those which can support the complete growth of the parasite long term, while producing a high yield of oocysts. While numerous cell lines have been reported as maintaining the parasite, there remain no options for maintaining the parasite for longer than a week. The current cell line of choice, HCT-8, can only maintain infection for three days. Recently, our lab has successfully cultivated C. parvum in the oesophageal cancer derived cell line COLO-680N, and can maintain infection for over a week. Given the success of this cell line, a panel of cancer derived cell lines were grown in the presence of C. parvum for one week, with development assessed with fluorescent microscopy and PCR. Four cell lines were used to establish cultures for three weeks. The lung adenocarcinoma cell line, HCC4006, gave the highest oocyst yield of 5.8 x 105 oocysts per mL, a nine fold return on the initial inoculum. In addition, to tackle the issue of long term oocyst production in vitro, a simple, low cost bioreactor system using the COLO-680N cell line was established which produced infectious oocysts for 13 weeks. This method of oocyst production will be used to establish further cell lines as long term culture platforms. Further work aims to characterise these cell lines to establish factors which promote parasite development, and to further improve yield with media supplementation.
  • Wicks, A. (2017). Pre-Treatment With BET Inhibitors Increases the Sensitivity of Cancer Cell Lines to Cisplatin and Oxaliplatin.
    Platinum compounds, such as cisplatin, carboplatin and oxaliplatin are utilised widely in therapeutic strategies against a range of malignancies, for instance Neuroblastoma and Ovarian cancer. These compounds elicit anticancer effects by damaging DNA via the formation of crosslinks with nitrogenous bases, leading to DNA damage responses and the induction of apoptotic signalling. Although these compounds may produce promising initial responses, the emergence and establishment of chemoresistant tumour cells can often result in tumour relapses and treatment failure. The mechanisms driving resistance to platinum compounds are numerous, and act via a diverse range of cellular processes, such as reducing uptake of the drug, increasing DNA repair activity, and reducing apoptotic signalling.
    There are also research efforts to elucidate the epigenetic modifications of resistant tumours, and the means in which these changes are recognised and transduced. The Bromodomain and Extra-Terminal domain (BET) family of proteins recognise acetylated histones, and regulate transcription via association with transcriptional co-activators and RNA polymerase II. BET family members, such as BRD4, are able to form 'super-enhancers', promoting oncogenic activity of genes such as MYC. Consequently, BET proteins have been identified as potential anticancer drug targets, prompting the development of BET inhibitors.
    This study investigated whether the BET inhibitors, JQ-1 and I-BET726, were able to increase the sensitivity of parental and oxaliplatin-resistant Neuroblastoma (UKF-NB3) and cisplatin-resistant Ovarian cancer cell lines (COLO-704, EFO-21 and EFO-27) to their respective platinum compounds. BET inhibitor pre-treatments, using the IC10 and IC50 concentrations of JQ-1 and I-BET726 as single-agents for each cell line, resulted in increased sensitivity to the platinum compounds, as determined by MTT cell viability assays. Increasing the pre-treatment incubation duration from 0hrs to 24hrs and 48hrs improved the response to platinum agents, and the same effect was observed with increasing pre-treatment dosage. In summary, these results suggest that BET inhibitors are able to increase the sensitivity of cancer cells to platinum chemotherapeutics.
  • Sanders, L. (2017). Investigation of Platinum Drug Mode of Action and Resistance Using Yeast and Human Neuroblastoma Cells As Model Systems.
    The platinum-based cytotoxic chemotherapeutics cisplatin, carboplatin, and oxaliplatin are widely used as anti-cancer drugs, but their efficacy is limited by the occurrence of resistance. The mechanisms of actions of these drugs as well as the mechanisms underlying resistance (formation) to platinum drugs remain incompletely understood. More knowledge is required to develop more effective therapies.

    In this study, we used yeast cells and neuroblastoma cell lines to investigate the mode of action and resistance to platinum drugs. First, we screened a Saccharomyces cerevisiae transcription factor heterozygous gene deletion library to identify genes that when deleted result in enhanced sensitivity to cisplatin, carboplatin and/or oxaliplatin. In addition, data on platinum drug sensitivity in yeast, derived from screening a yeast whole-genome homozygous deletion library, was extracted from the Yeast Fitness Database (http://fitdb.stanford.edu/). There was a substantial overlap in the genes (and related pathways) that determine sensitivity to the individual platinum drugs, but also considerable differences. Notably, cisplatin and carboplatin are anticipated to be more similar in their mode of action compared to oxaliplatin, but the yeast data did not entirely support this notion. Amoung the genes involved in response to platinum drugs, BDF1 (Bromodomain-containing factor 1) was identified as a novel gene which, when deleted, resulted in increased sensitivity to all three platinum drugs. Its re-expression reversed platinum drug sensitivity, confirming its role in determining the yeast cell response to platinum drugs. Notably, BET proteins (the human equivalents of Bdf1) are increasingly recognised as potential anti-cancer drug targets. Our data suggest that they may have a role in sensitising cancer cells to platinum drugs.

    Next, we investigated a unique panel of cell lines consisting of neuroblastoma cell lines UKF-NB-3 and UKF-NB-6 and their sub-lines with acquired resistance to cisplatin (UKF-NB-3rCDDP1000, UKF-NB-6rCDDP2000), oxaliplatin (UKF-NB-3rOXALI2000, UKF-NB6rOXALI4000), or carboplatin (UKF-NB3rCARBO2000, UKF-NB-6rCARBO2000). Adaption to platinum drugs was associated with changes in doubling times and cell morphology but there were no consistent patterns. This suggests that resistance mechanisms are complex and heterogeneous. The resistance phenotype was stable after cultivation of the resistant sub-lines for three months in the absence of drugs, indicating that resistance was not a consequence of the reversible enrichment of a pre-existing sub-population of cells, but due to a permanent, irreversible genomic change. This notion was supported by the determination of sensitivity profiles to cisplatin, carboplatin and oxaliplatin in the cell line panel. Both UKF-NB-3 and UKF-NB-6 parental lines exhibited sensitivity to all three platinum drugs, and the platinum drug-adapted parental cells displayed generally increased resistance, not just to the drug to which they were adapted, but to all three platinum drugs. However cisplatin- and carboplatin- resistant UKF-NB-3 cells displayed no cross-resistance to oxaliplatin, and oxaliplatin-resistant UKF-NB-3 cells displayed none to cisplatin and carboplatin. In contrast to the yeast data, this supports the notion that cisplatin and carboplatin are more similar in their mode of action than oxaliplatin.

    Finally, in a proteomics study, we compared the UKF-NB-3 and UKF-NB-6 cells with their cisplatin-resistant sub-lines to study acquired cisplatin resistance, and also investigated their responses to acute cisplatin treatment. The resulting data, together with previous proteomics studies that investigated acquired resistance in cisplatin-adapted neuroblastoma cell lines, suggested that, despite overlaps, the resistance mechanisms are heterogeneic and cell line-specific. This was also the case, comparing our cell lines only, for the acute cisplatin responses.

    In conclusion our data demonstrate that resistance formation to cisplatin is a complex and individual/cell line specific process. Further research will be required to enable a systems-level understanding of cisplatin resistance that can be translated into improved therapeutic approaches.
  • Ji, H. (2015). Fibrinolytic Regulation of Pulmonary Epithelial Sodium Channels: A Critical Review.
    Luminal fluid homeostasis in the respiratory system is crucial to maintain the gas-
    blood exchange in normal lungs and mucociliary clearance in the airways. Epithelial
    sodium channels (ENaC) govern ~70% of alveolar fluid clearance. Four ENaC subunits
    have been cloned, namely, ?, ?, ?, and ? ENaC subunits in mammalian cells. This
    critical review focuses on the expression and function of ENaC in human and murine
    lungs, and the post-translational regulation by fibrinolysins. Nebulized urokinase was
    intratracheally delivered for clinical models of lung injury with unknown mechanisms.
    The central hypothesis is that proteolytically cleaved ENaC channels composed of four
    subunits are essential pathways to maintain fluid homeostasis in the airspaces, and that
    fibrinolysins are potential pharmaceutical ENaC activators to resolve edema fluid. This
    hypothesis is strongly supported by our following observations: 1) ? ENaC is expressed
    in the apical membrane of human lung epithelial cells; 2) ? ENaC physically interacts
    with the other three ENaC counterparts; 3) the features of ??? ENaC channels are
    conferred by ? ENaC; 4) urokinase activates ENaC activity; 5) urokinase deficiency is
    associated with a markedly distressed pulmonary ENaC function in vivo; 6) ? ENaC is
    proteolytically cleaved by urokinase; 7) urokinase augments the density of opening
    channels at the cell surface; and 8) urokinase extends opening time of ENaC channels
    to the most extent. Our integrated publications laid the groundwork for an innovative
    concept of pulmonary transepithelial fluid clearance in both normal and diseased lungs.
  • Hanly, D. (2015). Investigating the Genomic Basis for Cancer Cell Response to Multi-AGC Kinase Inhibitor AT13148.
    The PI3K-AKT cellular growth and survival signalling pathway is commonly deregulated in a wide range of cancers. The novel candidate anticancer drug AT13148 is a multi-AGC kinase whose targets include PI3K-AKT signalling effectors, and it is currently in phase I clinical trial. Recently, growth inhibition and gene mutation data for this compound have been produced from the Genomics of Drug Sensitivity in Cancer (GDSC) cell line panel at the Wellcome Trust Sanger Institute. When bioinformatics pharmacogenomic analyses were performed for common cancer genes, PTEN mutations were statistically significantly associated with AT13148 sensitivity, whereas KRAS mutations were significantly associated with resistance. Amplifications of KIT and PDGFRA, both individually and together with KDR were associated with potent AT13148 sensitivity. Whole exome pharmacogenomic analyses also revealed a further fifteen genes mutationally associated with sensitivity to AT13148, including VHL mutations predominantly in kidney cancer cell lines and rare mutations in CETN3, NOP10 and CLDND2 occurring in highly sensitive cell lines. IC50 comparison analyses between AT13148 and other drugs included on the GDSC database, SRC inhibitors Dasatinib, AZD-0530 and A-770041 gave strong IC50 correlations with AT13148 in kidney cancer cell lines. Future genomic analyses of AT13148 GDSC data should investigate the mutational characteristics of AT13148-sensitive PTEN mutated cell lines and further examine the genomic features of AT13148-responsive kidney cancer cell lines. Transcriptomics analysis approaches should also be developed and integrated into these investigations. Laboratory experiments should also be developed to test the pharmacogenomic associations with AT13148 response predicted in this study using a distinct set of cell lines. Results of such experiments, if consistent with predictions, may potentially assist with patient stratification for phase II clinical trial of AT13148.
  • Aderemi, A. (2015). Characterisation of Drug-Resistant Neuroblastoma Cell Lines.
    Resistance acquisition to chemotherapy is a major cause of treatment failure in high-risk neuroblastoma. This report focused on the use of drug-adapted neuroblastoma cell lines as pre-clinical models of acquired drug resistance. Results indicated intact cell MALDI-ToF mass spectrometry (MS) fingerprinting to be a promising technique for the authentication of drug-adapted cancer cell lines that cannot be discriminated from their parental cell lines by standard methods like short tandem repeat analysis, but further optimisation is required. MS fingerprinting and the determination of platinum drug-resistance profiles in 10 single cell-derived clonal sub-lines of the docetaxel-adapted UKF-NB-3 sub-line UKF-NB-3rDOCE10 revealed a noticeable heterogeneity. Further studies need to be performed in other cell line models and including additional anti-cancer agents to learn more about the heterogeneity in drug-resistant cancer cell populations in comparison to the respective parental cancer cell populations. Newly synthesised platinum- and palladium-based compounds displayed encouraging anti-cancer efficacy in the neuroblastoma cell line UKF-NB-3 and its sub-lines adapted to cisplatin (UKF-NB-3rCDDP1000), carboplatin (UKF-NB-3rCARBO2000), or oxaliplatin (UKF-NB-3rOXALI2000) warranting further studies in additional cell lines and animal experiments. 1H-NMR-based metabonomics characterisation of UKF-NB-3, UKF-NB-3rCDDP1000, UKF-NB-3rCARBO2000, and UKF-NB-3rOXALI2000 revealed remarkable differences and did, thus, not suggest that a certain sub-population of pre-existing cancer cells was selected during UKF-NB-3 cell adaptation to these closely related platinum drugs. Further research will have to show the variability of the resistance formation process when the same cell line is adapted to the same drug in parallel independent experiments. In conclusion, the data presented here provide initial information for the further use of drug-adapted cancer cell lines.
  • Kajewole, D. (2014). Drug Sensitivity Profiles in Neuroblastoma Cells With Acquired Resistance to Stabilising Tubulin Binding Agents.
    Tubulin-binding agents either inhibit microtubule formation (destabilising agents) or degradation (stabilising agents) thereby interfering with the dynamics required for microtubule functions resulting in cell cycle disruption and cell death. In this project, acquired resistance of neuroblastoma cell lines to tubulin-binding agents was studied.
    We investigated the resistance profiles of 10 clonal sub-lines of the docetaxel-resistant neuroblastoma cell line, UKF-NB-3rDOCE10 and 4 epothilone B-resistant UKF-NB-3 sub-lines that had been established in independent experiments against the tubulin-binding agents: combretastatin A4, 2-methoxyestradiol (destabilising agents, target the colchicine domain), vincristine, vinblastine (destabilising agents, target the vinca-domain), docetaxel, and epothilone B (stabilising agents, bind at the taxoid domain). Remarkable differences were detected in the resistance profiles of the UKF-NB-3rDOCE10 sub-clones and the epothilone B-resistant UKF-NB-3 sub-lines.
    Further, we started to establish a protocol for the standardised adaptation of cancer cells to anti-cancer drugs. In uneven weeks, 40000 UKF-NB-3 cells per cm2 were treated with the IC50 concentration of the selected drug. In even weeks, 4000 UKF-NB-3 cells per cm2 were allowed to grow in the absence of drug. We started to establish 10 cabazitaxel-, 10 epothilone B- and 10 paclitaxel-resistant UKF-NB-3 sub-lines by this approach. The paclitaxel-treated sub-lines displayed a trend towards decreased paclitaxel sensitivity relative to UKF-NB-3 after four and eight weeks, while the epothilone B- and cabazitaxel-treated sub-lines displayed a trend towards enhanced sensitivity to the respective drugs.
    In conclusion, our data suggest 1) that the repeated adaptation of a cell line to the same drug results in heterogenic sub-lines, 2) that the adaptation of a cancer cell line to a drug results in heterogeneity in the resulting resistant sub-line (as opposed to the selection of a pre-existing drug-resistant sub-population) and 3) that exposure of a cell population to different drugs with a similar mechanism of action may result in varying outcomes.
Last updated