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  • 1.
    Adori, Csaba
    et al.
    Karolinska Inst, Dept Neurosci, Retzius Lab, Retzius Vag 8, S-17177 Stockholm, Sweden.
    Barde, Swapnali
    Karolinska Inst, Dept Neurosci, Retzius Lab, Retzius Vag 8, S-17177 Stockholm, Sweden.
    Vas, Szilvia
    Semmelweis Univ, Dept Pharmacodynam, Nagyvarad Ter 4, H-1089 Budapest, Hungary; Hungarian Acad Sci, Neuropsychopharmacol & Neurochem Res Grp, Nagyvarad Ter 4, H-1089 Budapest, Hungary.
    Ebner, Karl
    Leopold Franzens Univ Innsbruck, CMBI, Inst Pharm, Dept Pharmacol & Toxicol, Innrain 80-82-3, A-6020 Innsbruck, Austria.
    Su, Jie
    Karolinska Inst, Dept Physiol & Pharmacol, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden.
    Svensson, Camilla
    Karolinska Inst, Dept Physiol & Pharmacol, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden.
    Mathé, Aleksander A
    Karolinska Inst, Sect Psychiat, Dept Clin Neurosci, Tomtebodavagen 18A, S-17177 Stockholm, Sweden.
    Singewald, Nicolas
    Leopold Franzens Univ Innsbruck, CMBI, Inst Pharm, Dept Pharmacol & Toxicol, Innrain 80-82-3, A-6020 Innsbruck, Austria.
    Reinscheid, Rainer R
    Univ Calif Irvine, Dept Pharmaceut Sci, Irvine, CA 92697 USA.
    Uhlén, Mathias
    Karolinska Inst, Dept Neurosci, Sci Life Lab, S-17165 Stockholm, Sweden; Royal Inst Technol, Albanova Univ Ctr, Sci Life Lab, S-17165 Stockholm, Sweden.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bagdy, György
    Semmelweis Univ, Dept Pharmacodynam, Nagyvarad Ter 4, H-1089 Budapest, Hungary; Hungarian Acad Sci, Neuropsychopharmacol & Neurochem Res Grp, Nagyvarad Ter 4, H-1089 Budapest, Hungary.
    Hökfelt, Tomas
    Karolinska Inst, Dept Neurosci, Retzius Lab, Retzius Vag 8, S-17177 Stockholm, Sweden.
    Exploring the role of neuropeptide S in the regulation of arousal: a functional anatomical study.2016In: Brain Structure and Function, ISSN 1863-2653, E-ISSN 1863-2661, Vol. 221, no 7, p. 3521-3546Article in journal (Refereed)
    Abstract [en]

    Neuropeptide S (NPS) is a regulatory peptide expressed by limited number of neurons in the brainstem. The simultaneous anxiolytic and arousal-promoting effect of NPS suggests an involvement in mood control and vigilance, making the NPS-NPS receptor system an interesting potential drug target. Here we examined, in detail, the distribution of NPS-immunoreactive (IR) fiber arborizations in brain regions of rat known to be involved in the regulation of sleep and arousal. Such nerve terminals were frequently apposed to GABAergic/galaninergic neurons in the ventro-lateral preoptic area (VLPO) and to tyrosine hydroxylase-IR neurons in all hypothalamic/thalamic dopamine cell groups. Then we applied the single platform-on-water (mainly REM) sleep deprivation method to study the functional role of NPS in the regulation of arousal. Of the three pontine NPS cell clusters, the NPS transcript levels were increased only in the peri-coerulear group in sleep-deprived animals, but not in stress controls. The density of NPS-IR fibers was significantly decreased in the median preoptic nucleus-VLPO region after the sleep deprivation, while radioimmunoassay and mass spectrometry measurements showed a parallel increase of NPS in the anterior hypothalamus. The expression of the NPS receptor was, however, not altered in the VLPO-region. The present results suggest a selective activation of one of the three NPS-expressing neuron clusters as well as release of NPS in distinct forebrain regions after sleep deprivation. Taken together, our results emphasize a role of the peri-coerulear cluster in the modulation of arousal, and the importance of preoptic area for the action of NPS on arousal and sleep.

  • 2.
    Aftab, Obaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Towards High-Throughput Phenotypic and Systemic Profiling of in vitro Growing Cell Populations using Label-Free Microscopy and Spectroscopy: Applications in Cancer Pharmacology2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.

    List of papers
    1. Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter
    Open this publication in new window or tab >>Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter
    Show others...
    2014 (English)In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 10, no 1, p. 57-69Article in journal (Refereed) Published
    Abstract [en]

    Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.

    Keywords
    phase-contrast microscopy, automated microscopy, vesicle detection, autophagy, image processing
    National Category
    Clinical Medicine
    Identifiers
    urn:nbn:se:uu:diva-216046 (URN)10.4161/auto.26678 (DOI)000328812400006 ()
    Conference
    High Content Anlaysis
    Available from: 2014-01-20 Created: 2014-01-17 Last updated: 2017-12-06Bibliographically approved
    2. Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing
    Open this publication in new window or tab >>Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing
    Show others...
    2014 (English)In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 19, no 9, p. 1411-1418Article in journal (Refereed) Published
    Abstract [en]

    Label free time-lapse microscopy has opened a new avenue to the study of time evolving events in living cells. When combined with automated image analysis it provides a powerful tool that enables automated large-scale spatiotemporal quantification at the cell population level. Very few attempts, however, have been reported regarding the design of image analysis algorithms dedicated to the detection of apoptotic cells in such time-lapse microscopy images. In particular, none of the reported attempts is based on sufficiently fast signal processing algorithms to enable large-scale detection of apoptosis within hours/days without access to high-end computers. Here we show that it is indeed possible to successfully detect chemically induced apoptosis by applying a two-dimensional linear matched filter tailored to the detection of objects with the typical features of an apoptotic cell in phase-contrast images. First a set of recorded computational detections of apoptosis was validated by comparison with apoptosis specific caspase activity readouts obtained via a fluorescence based assay. Then a large screen encompassing 2,866 drug like compounds was performed using the human colorectal carcinoma cell line HCT116. In addition to many well known inducers (positive controls) the screening resulted in the detection of two compounds here reported for the first time to induce apoptosis.

    Keywords
    Apoptosis, high throughput screening, cancer
    National Category
    Cancer and Oncology
    Research subject
    Bioinformatics
    Identifiers
    urn:nbn:se:uu:diva-229069 (URN)10.1007/s10495-014-1009-9 (DOI)000340518000010 ()
    Funder
    Swedish Society for Medical Research (SSMF)
    Available from: 2014-07-29 Created: 2014-07-29 Last updated: 2018-01-09Bibliographically approved
    3. Detection of cell aggregation and altered cell viability by automated label-free video microscopy: A promising alternative to endpoint viability assays in high throughput screening
    Open this publication in new window or tab >>Detection of cell aggregation and altered cell viability by automated label-free video microscopy: A promising alternative to endpoint viability assays in high throughput screening
    Show others...
    2015 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, no 3, p. 372-381Article in journal (Refereed) Published
    Abstract [en]

    Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.

    Keywords
    time-lapse microscopy, video microscopy, phase contrast microscopy, differentially time evolving morphologies, high throughput screening (HTS), cell aggregation, PDGFR signalling.
    National Category
    Bioinformatics (Computational Biology) Social and Clinical Pharmacy
    Research subject
    Bioinformatics; Clinical Pharmacology
    Identifiers
    urn:nbn:se:uu:diva-234561 (URN)10.1177/1087057114562158 (DOI)000350310000007 ()25520371 (PubMedID)
    Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2018-01-11Bibliographically approved
    4. Label free quantification of time evolving morphologies using time-lapse video microscopy enables identity control of cell lines and discovery of chemically induced differential activity in iso-genic cell line pairs
    Open this publication in new window or tab >>Label free quantification of time evolving morphologies using time-lapse video microscopy enables identity control of cell lines and discovery of chemically induced differential activity in iso-genic cell line pairs
    Show others...
    2015 (English)In: Chemometrics and Intelligent Laboratory Systems, ISSN 0169-7439, E-ISSN 1873-3239, Vol. 141, p. 24-32Article in journal (Refereed) Published
    Abstract [en]

    Label free time-lapse video microscopy based monitoring of time evolving cell population morphology has potential to offer a simple and cost effective method for identity control of cell lines. Such morphology monitoring also has potential to offer discovery of chemically induced differential changes between pairs of cell lines of interest, for example where one in a pair of cell lines is normal/sensitive and the other malignant/resistant. A new simple algorithm, pixel histogram hierarchy comparison (PHHC), for comparison of time evolving morphologies (TEM) in phase contrast time-lapse microscopy movies was applied to a set of 10 different cell lines and three different iso-genic colon cancer cell line pairs, each pair being genetically identical except for a single mutation. PHHC quantifies differences in morphology by comparing pixel histogram intensities at six different resolutions. Unsupervised clustering and machine learning based classification methods were found to accurately identify cell lines, including their respective iso-genic variants, through time-evolving morphology. Using this experimental setting, drugs with differential activity in iso-genic cell line pairs were likewise identified. Thus, this is a cost effective and expedient alternative to conventional molecular profiling techniques and might be useful as part of the quality control in research incorporating cell line models, e.g. in any cell/tumor biology or toxicology project involving drug/agent differential activity in pairs of cell line models.

    Keywords
    Time evolving morphology, quality control, iso-genic cell line, cancer pharmacology, time-lapse microsopcy, video microscopy
    National Category
    Computer Sciences Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-234563 (URN)10.1103/PhysRevC.91.024602 (DOI)000350096200003 ()
    Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2018-01-11Bibliographically approved
    5. NMR spectroscopy based metabolic profiling of drug induced changes in vitro can discriminate between pharmacological classes
    Open this publication in new window or tab >>NMR spectroscopy based metabolic profiling of drug induced changes in vitro can discriminate between pharmacological classes
    Show others...
    2014 (English)In: Journal of chemical information and modeling, ISSN 1549-9596, Vol. 54, no 11, p. 3251-3258Article in journal (Refereed) Published
    Abstract [en]

    Drug induced changes in mammalian cell line models have already been extensively profiled at the systemic mRNA level and subsequently used to suggest mechanisms of action for new substances as well as to support drug repurposing, i.e. identifying new potential indications for drugs already licensed for other pharmacotherapy settings. The seminal work in this field, which includes a large database and computational algorithms for pattern matching, is known as the “Connectivity Map” (CMap). The potential of similar exercises at the metabolite level is, however, still largely unexplored. Only recently the first high throughput metabolomic assay pilot study was published, involving screening of metabolic response to a set of 56 kinase inhibitors in a 96-well format. Here we report results from a separately developed metabolic profiling assay, which leverages 1H NMR spectroscopy to the quantification of metabolic changes in the HCT116 colorectal cancer cell line, in response to each of 26 compounds. These agents are distributed across 12 different pharmacological classes covering a broad spectrum of bioactivity. Differential metabolic profiles, inferred from multivariate spectral analysis of 18 spectral bins, allowed clustering of most tested drugs according to their respective pharmacological class. A more advanced supervised analysis, involving one multivariate scattering matrix per pharmacological class and using only 3 spectral bins (three metabolites), showed even more distinct pharmacology-related cluster formations. In conclusion, this kind of relatively fast and inexpensive profiling seems to provide a promising alternative to that afforded by mRNA expression analysis, which is relatively slow and costly. As also indicated by the present pilot study, the resulting metabolic profiles do not seem to provide as information rich signatures as those obtained using systemic mRNA profiling, but the methodology holds strong promise for significant refinement.

    National Category
    Cancer and Oncology
    Identifiers
    urn:nbn:se:uu:diva-234564 (URN)10.1021/ci500502f (DOI)000345551000021 ()25321343 (PubMedID)
    Available from: 2014-10-21 Created: 2014-10-21 Last updated: 2015-02-03Bibliographically approved
  • 3.
    Aftab, Obaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Engskog, Mikael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Haglöf, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Elmsjö, Albert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Hammerling, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    NMR spectroscopy based metabolic profiling of drug induced changes in vitro can discriminate between pharmacological classes2014In: Journal of chemical information and modeling, ISSN 1549-9596, Vol. 54, no 11, p. 3251-3258Article in journal (Refereed)
    Abstract [en]

    Drug induced changes in mammalian cell line models have already been extensively profiled at the systemic mRNA level and subsequently used to suggest mechanisms of action for new substances as well as to support drug repurposing, i.e. identifying new potential indications for drugs already licensed for other pharmacotherapy settings. The seminal work in this field, which includes a large database and computational algorithms for pattern matching, is known as the “Connectivity Map” (CMap). The potential of similar exercises at the metabolite level is, however, still largely unexplored. Only recently the first high throughput metabolomic assay pilot study was published, involving screening of metabolic response to a set of 56 kinase inhibitors in a 96-well format. Here we report results from a separately developed metabolic profiling assay, which leverages 1H NMR spectroscopy to the quantification of metabolic changes in the HCT116 colorectal cancer cell line, in response to each of 26 compounds. These agents are distributed across 12 different pharmacological classes covering a broad spectrum of bioactivity. Differential metabolic profiles, inferred from multivariate spectral analysis of 18 spectral bins, allowed clustering of most tested drugs according to their respective pharmacological class. A more advanced supervised analysis, involving one multivariate scattering matrix per pharmacological class and using only 3 spectral bins (three metabolites), showed even more distinct pharmacology-related cluster formations. In conclusion, this kind of relatively fast and inexpensive profiling seems to provide a promising alternative to that afforded by mRNA expression analysis, which is relatively slow and costly. As also indicated by the present pilot study, the resulting metabolic profiles do not seem to provide as information rich signatures as those obtained using systemic mRNA profiling, but the methodology holds strong promise for significant refinement.

  • 4.
    Aftab, Obaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Hammerling, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Detection of cell aggregation and altered cell viability by automated label-free video microscopy: A promising alternative to endpoint viability assays in high throughput screening2015In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, no 3, p. 372-381Article in journal (Refereed)
    Abstract [en]

    Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.

  • 5.
    Aftab, Obaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Hassan, Saadia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Hammerling, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Label free quantification of time evolving morphologies using time-lapse video microscopy enables identity control of cell lines and discovery of chemically induced differential activity in iso-genic cell line pairs2015In: Chemometrics and Intelligent Laboratory Systems, ISSN 0169-7439, E-ISSN 1873-3239, Vol. 141, p. 24-32Article in journal (Refereed)
    Abstract [en]

    Label free time-lapse video microscopy based monitoring of time evolving cell population morphology has potential to offer a simple and cost effective method for identity control of cell lines. Such morphology monitoring also has potential to offer discovery of chemically induced differential changes between pairs of cell lines of interest, for example where one in a pair of cell lines is normal/sensitive and the other malignant/resistant. A new simple algorithm, pixel histogram hierarchy comparison (PHHC), for comparison of time evolving morphologies (TEM) in phase contrast time-lapse microscopy movies was applied to a set of 10 different cell lines and three different iso-genic colon cancer cell line pairs, each pair being genetically identical except for a single mutation. PHHC quantifies differences in morphology by comparing pixel histogram intensities at six different resolutions. Unsupervised clustering and machine learning based classification methods were found to accurately identify cell lines, including their respective iso-genic variants, through time-evolving morphology. Using this experimental setting, drugs with differential activity in iso-genic cell line pairs were likewise identified. Thus, this is a cost effective and expedient alternative to conventional molecular profiling techniques and might be useful as part of the quality control in research incorporating cell line models, e.g. in any cell/tumor biology or toxicology project involving drug/agent differential activity in pairs of cell line models.

  • 6.
    Aftab, Obaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Zhang, Xiaonan
    De Milito, Angelo
    Hammerling, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Linder, Stig
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter2014In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 10, no 1, p. 57-69Article in journal (Refereed)
    Abstract [en]

    Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.

  • 7.
    Aftab, Obaid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nazir, Madiha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Hammerling, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing2014In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 19, no 9, p. 1411-1418Article in journal (Refereed)
    Abstract [en]

    Label free time-lapse microscopy has opened a new avenue to the study of time evolving events in living cells. When combined with automated image analysis it provides a powerful tool that enables automated large-scale spatiotemporal quantification at the cell population level. Very few attempts, however, have been reported regarding the design of image analysis algorithms dedicated to the detection of apoptotic cells in such time-lapse microscopy images. In particular, none of the reported attempts is based on sufficiently fast signal processing algorithms to enable large-scale detection of apoptosis within hours/days without access to high-end computers. Here we show that it is indeed possible to successfully detect chemically induced apoptosis by applying a two-dimensional linear matched filter tailored to the detection of objects with the typical features of an apoptotic cell in phase-contrast images. First a set of recorded computational detections of apoptosis was validated by comparison with apoptosis specific caspase activity readouts obtained via a fluorescence based assay. Then a large screen encompassing 2,866 drug like compounds was performed using the human colorectal carcinoma cell line HCT116. In addition to many well known inducers (positive controls) the screening resulted in the detection of two compounds here reported for the first time to induce apoptosis.

  • 8.
    Ahnoff, Martin
    et al.
    Univ Gothenburg, Dept Chem Mol Biol, SE-41296 Gothenburg, Sweden.;Denator AB, Gothenburg, Sweden..
    Cazares, Lisa H.
    US Army Med Res Inst Infect Dis, Mol & Translat Sci, Frederick, MD 21702 USA.;US Army Med Res & Mat Command, DoD Biotechnol High Performance Comp Software App, Telemed & Adv Technol Res Ctr, Ft Detrick, MD 21702 USA..
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Denator AB, Gothenburg, Sweden.;Uppsala Univ, Dept Med Sci Canc Pharmacol & Computat Med, Uppsala, Sweden..
    Thermal inactivation of enzymes and pathogens in biosamples for MS analysis2015In: Bioanalysis, ISSN 1757-6180, E-ISSN 1757-6199, Vol. 7, no 15, p. 1885-1899Article, review/survey (Refereed)
    Abstract [en]

    Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (approximate to 1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

  • 9.
    Almandoz-Gil, Leire
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Welander, Hedvig
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ihse, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Khoonsari, Payam Emami
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Musunuri, Sravani
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lendel, Christofer
    KTH Royal Inst Technol, Dept Chem, Stockholm, Sweden.
    Sigvardson, Jessica
    BioArctic AB, Stockholm, Sweden.
    Karlsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bergström, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Corrigendum to “Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways” [Free Rad. Biol. Med. (2017) 421–431]2018In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 117, p. 258-258Article in journal (Other academic)
  • 10.
    Almandoz-Gil, Leire
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Welander, Hedvig
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ihse, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Khoonsari, Payam Emami
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Musunuri, Sravani
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Lendel, Christofer
    KTH, Royal Institute of Technology, Sweden.
    Sigvardson, Jessica
    BioArctic AB, Sweden.
    Karlsson, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Applied Materials Sciences.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bergström, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways2017In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 110, p. 421-431Article in journal (Refereed)
    Abstract [en]

    Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.

  • 11.
    Alvarsson, Jonathan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Eklund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Carlsson, Lars
    AstraZeneca R&D.
    Spjuth, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wikberg, Jarl E. S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Benchmarking Study of Parameter Variation When Using Signature Fingerprints Together with Support Vector Machines2014In: Journal of Chemical Information and Modeling, ISSN 1549-9596, Vol. 54, no 11, p. 3211-3217Article in journal (Refereed)
    Abstract [en]

    QSAR modeling using molecular signatures and support vector machines with a radial basis function is increasingly used for virtual screening in the drug discovery field. This method has three free parameters: C, ?, and signature height. C is a penalty parameter that limits overfitting, ? controls the width of the radial basis function kernel, and the signature height determines how much of the molecule is described by each atom signature. Determination of optimal values for these parameters is time-consuming. Good default values could therefore save considerable computational cost. The goal of this project was to investigate whether such default values could be found by using seven public QSAR data sets spanning a wide range of end points and using both a bit version and a count version of the molecular signatures. On the basis of the experiments performed, we recommend a parameter set of heights 0 to 2 for the count version of the signature fingerprints and heights 0 to 3 for the bit version. These are in combination with a support vector machine using C in the range of 1 to 100 and gamma in the range of 0.001 to 0.1. When data sets are small or longer run times are not a problem, then there is reason to consider the addition of height 3 to the count fingerprint and a wider grid search. However, marked improvements should not be expected.

  • 12.
    Alvarsson, Jonathan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lampa, Samuel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Schaal, Wesley
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Wikberg, Jarl E. S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Spjuth, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Large-scale ligand-based predictive modelling using support vector machines2016In: Journal of Cheminformatics, ISSN 1758-2946, E-ISSN 1758-2946, Vol. 8, article id 39Article in journal (Refereed)
    Abstract [en]

    The increasing size of datasets in drug discovery makes it challenging to build robust and accurate predictive models within a reasonable amount of time. In order to investigate the effect of dataset sizes on predictive performance and modelling time, ligand-based regression models were trained on open datasets of varying sizes of up to 1.2 million chemical structures. For modelling, two implementations of support vector machines (SVM) were used. Chemical structures were described by the signatures molecular descriptor. Results showed that for the larger datasets, the LIBLINEAR SVM implementation performed on par with the well-established libsvm with a radial basis function kernel, but with dramatically less time for model building even on modest computer resources. Using a non-linear kernel proved to be infeasible for large data sizes, even with substantial computational resources on a computer cluster. To deploy the resulting models, we extended the Bioclipse decision support framework to support models from LIBLINEAR and made our models of logD and solubility available from within Bioclipse.

  • 13.
    Banduseela, Varuna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology.
    Chen, Yi-wen
    Göransson Kultima, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Norman, Holly
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology. Department of Medicine, University of Wisconsin, Madison, Wisconsin.
    Aare, Sudhakar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology.
    Radell, Peter
    Eriksson, Lars
    Hoffman, Eric
    Larsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology. Department of Biobehavioral Health, The Pennsylvania State University, University Park, Pennsylvania.
    Impaired autophagy, chaperone expression, and protein synthesis in response to critical illness interventions in porcine skeletal muscle2013In: Physiological Genomics, ISSN 1094-8341, E-ISSN 1531-2267, Vol. 45, no 12, p. 477-486Article in journal (Refereed)
    Abstract [en]

    Critical illness myopathy (CIM) is characterized by a preferential loss of the motor protein myosin, muscle wasting, and impaired muscle function in critically ill intensive care unit (ICU) patients. CIM is associated with severe morbidity and mortality and has a significant negative socioeconomic effect. Neuromuscular blocking agents, corticosteroids, sepsis, mechanical ventilation, and immobilization have been implicated as important risk factors, but the causal relationship between CIM and the risk factors has not been established. A porcine ICU model has been used to determine the immediate molecular and cellular cascades that may contribute to the pathogenesis prior to myosin loss and extensive muscle wasting. Expression profiles have been compared between pigs exposed to the ICU interventions, i.e., mechanically ventilated, sedated, and immobilized for 5 days, with pigs exposed to critical illness interventions, i.e., neuromuscular blocking agents, corticosteroids, and induced sepsis in addition to the ICU interventions for 5 days. Impaired autophagy as well as impaired chaperone expression and protein synthesis were observed in the skeletal muscle in response to critical illness interventions. A novel finding in this study is impaired core autophagy machinery in response to critical illness interventions, which when in concert with downregulated chaperone expression and protein synthesis may collectively affect the proteostasis in skeletal muscle and may exacerbate the disease progression in CIM.

  • 14. Berenjian, Saideh
    et al.
    Hu, Kefei
    Abedi-Valugerdi, Manuchehr
    Hassan, Moustapha
    Hassan, Sadia Bashir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Morein, Bror
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    The nanoparticulate Quillaja saponin KGI exerts anti-proliferative eff ects by down-regulation of cell cycle molecules in U937 and HL-60 human leukemia cells2014In: Leukemia and Lymphoma, ISSN 1042-8194, E-ISSN 1029-2403, Vol. 55, no 7, p. 1618-1624Article in journal (Refereed)
    Abstract [en]

    Cancer cells are characterized by uncontrolled replication involving loss of control of cyclin dependent kinases (CDKs) and cyclins, and by abolished differentiation. In this study we introduce KGI, which is a nanoparticle with a Quillaja saponin as an active molecule. By the use of RNA array analysis and confirmation at the protein level, we show that KGI affects myeloid leukemia cells (in particular, the U937 monoblast cancer cell) by the following mechanisms: (A) ceasing cell replication via proteasome degradation, (B) down-regulation of key molecules at check points between G1/S and G2/M phases, (C) reduction of thymidine kinase activity, followed by (D) exit to differentiation and production of interleukin-8 (IL-8), eventually leading to apoptosis. Leukemia cell lines (U937 and HL-60 cells) were exposed to KGI for 8 h, after which the drug was removed. The cancer cells did not revert to replication over the following 10 days. Thus our findings suggest that the nanoparticle KGI inhibits proliferation and promotes differentiation in leukemic cells by interfering with the cell cycle process.

  • 15.
    Berg, L.
    et al.
    Karolinska Inst, S-10401 Stockholm, Sweden..
    Sundström, Y.
    Karolinska Inst, S-10401 Stockholm, Sweden..
    Aftab, Obaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bergqvist, F.
    Karolinska Inst, S-10401 Stockholm, Sweden..
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Sundström, M.
    Karolinska Inst, S-10401 Stockholm, Sweden..
    Ossipova, E.
    Karolinska Inst, S-10401 Stockholm, Sweden..
    Lengqvist, J.
    Karolinska Inst, S-10401 Stockholm, Sweden..
    Jakobsson, P-J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Rubin, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Characterizing the effects of epigenetic regulation in assays using peripheral blood mononuclear cells from patients with inflammatory diseases2016In: Scandinavian Journal of Rheumatology, ISSN 0300-9742, E-ISSN 1502-7732, Vol. 45, p. 44-45Article in journal (Other academic)
  • 16.
    Birgisson, Helgi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Colorectal Surgery.
    Edlund, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Wallin, Ulrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Colorectal Surgery.
    Påhlman, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Colorectal Surgery.
    Kultima, Hanna Göransson
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mayrhofer, Markus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Isaksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Glimelius, Bengt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Sundström, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Microsatellite instability and mutations in BRAF and KRAS are significant predictors of disseminated disease in colon cancer2015In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 15, article id 125Article in journal (Refereed)
    Abstract [en]

    Background: Molecular alterations are well studied in colon cancer, however there is still need for an improved understanding of their prognostic impact. This study aims to characterize colon cancer with regard to KRAS, BRAF, and PIK3CA mutations, microsatellite instability (MSI), and average DNA copy number, in connection with tumour dissemination and recurrence in patients with colon cancer. Methods: Disease stage II-IV colon cancer patients (n = 121) were selected. KRAS, BRAF, and PIK3CA mutation status was assessed by pyrosequencing and MSI was determined by analysis of mononucleotide repeat markers. Genome-wide average DNA copy number and allelic imbalance was evaluated by SNP array analysis. Results: Patients with mutated KRAS were more likely to experience disease dissemination (OR 2.75; 95% CI 1.28-6.04), whereas the opposite was observed for patients with BRAF mutation (OR 0.34; 95% 0.14-0.81) or MSI (OR 0.24; 95% 0.09-0.64). Also in the subset of patients with stage II-III disease, both MSI (OR 0.29; 95% 0.10-0.86) and BRAF mutation (OR 0.32; 95% 0.16-0.91) were related to lower risk of distant recurrence. However, average DNA copy number and PIK3CA mutations were not associated with disease dissemination. Conclusions: The present study revealed that tumour dissemination is less likely to occur in colon cancer patients with MSI and BRAF mutation, whereas the presence of a KRAS mutation increases the likelihood of disseminated disease.

  • 17.
    Blom, Kristin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Alvarsson, Jonathan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Ex Vivo Assessment of Drug Activity in Patient Tumor Cells as a Basis for Tailored Cancer Therapy2016In: JALA, ISSN 2211-0682, Vol. 21, no 1, p. 178-187Article in journal (Refereed)
    Abstract [en]

    Although medical cancer treatment has improved during the past decades, it is difficult to choose between several first-line treatments supposed to be equally active in the diagnostic group. It is even more difficult to select a treatment after the standard protocols have failed. Any guidance for selection of the most effective treatment is valuable at these critical stages. We describe the principles and procedures for ex vivo assessment of drug activity in tumor cells from patients as a basis for tailored cancer treatment. Patient tumor cells are assayed for cytotoxicity with a panel of drugs. Acoustic drug dispensing provides great flexibility in the selection of drugs for testing; currently, up to 80 compounds and/or combinations thereof may be tested for each patient. Drug response predictions are obtained by classification using an empirical model based on historical responses for the diagnosis. The laboratory workflow is supported by an integrated system that enables rapid analysis and automatic generation of the clinical referral response.

  • 18.
    Blom, Kristin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Predictive Value of Ex Vivo Chemosensitivity Assays for Individualized Cancer Chemotherapy: A Meta-Analysis2017In: SLAS TECHNOLOGY, ISSN 2472-6303, Vol. 22, no 3, p. 306-314Article in journal (Refereed)
    Abstract [en]

    Current treatment strategies for chemotherapy of cancer patients were developed to benefit groups of patients with similar clinical characteristics. In practice, response is very heterogeneous between individual patients within these groups. Precision medicine can be viewed as the development toward a more fine-grained treatment stratification than what is currently in use. Cell-based drug sensitivity testing is one of several options for individualized cancer treatment available today, although it has not yet reached widespread clinical use. We present an up-to-date literature meta-analysis on the predictive value of ex vivo chemosensitivity assays for individualized cancer chemotherapy and discuss their current clinical value and possible future developments.

  • 19.
    Blom, Kristin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Senkowski, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Berglund, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Rubin, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Parrow, Vendela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Loskog, Angelica
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    The anticancer effect of mebendazole may be due to M1 monocyte/macrophage activation via ERK1/2 and TLR8-dependent inflammasome activation2017In: Immunopharmacology and immunotoxicology, ISSN 0892-3973, E-ISSN 1532-2513, Vol. 39, no 4, p. 199-210Article in journal (Refereed)
    Abstract [en]

    Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1 beta and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1 beta secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1 release. MBZ-induced IL-1 release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ's anticancer activity observed in animal tumor models and in the clinic.

  • 20. Brnjic, Slavica
    et al.
    Mazurkiewicz, Magdalena
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Sun, Chao
    Zhang, Xiaonan
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    D'Arcy, Padraig
    Linder, Stig
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Induction of Tumor Cell Apoptosis by a Proteasome Deubiquitinase Inhibitor Is Associated with Oxidative Stress2014In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 21, no 17, p. 2271-2285Article in journal (Refereed)
    Abstract [en]

    Aims: b-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition. Results: We report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of alpha-subunit of eukaryotic initiation factor 2 phosphorylation. Innovation: The findings establish that different modes of proteasome inhibition result in distinct cellular responses, a finding of potential therapeutic importance. Conclusion: Our data show that enhanced oxidative stress and ER stress are major determinants of the strong apoptotic response elicited by the 19S DUB inhibitor b-AP15. Antioxid. Redox Signal. 21, 2271-2285.

  • 21. Brunberg, E.
    et al.
    Jensen, P.
    Isaksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Keeling, L. J.
    Brain gene expression differences are associated with abnormal tail biting behavior in pigs2013In: Genes, Brain and Behavior, ISSN 1601-1848, E-ISSN 1601-183X, Vol. 12, no 2, p. 275-281Article in journal (Refereed)
    Abstract [en]

    Knowledge about gene expression in animals involved in abnormal behaviors can contribute to the understanding of underlying biological mechanisms. This study aimed to explore the motivational background to tail biting, an abnormal injurious behavior and severe welfare problem in pig production. Affymetrix microarrays were used to investigate gene expression differences in the hypothalamus and prefrontal cortex of pigs performing tail biting, pigs receiving bites to the tail and neutral pigs who were not involved in the behavior. In the hypothalamus, 32 transcripts were differentially expressed (P<0.05) when tail biters were compared with neutral pigs, 130 when comparing receiver pigs with neutrals, and two when tail biters were compared with receivers. In the prefrontal cortex, seven transcripts were differently expressed in tail biters when compared with neutrals, seven in receivers vs. neutrals and none in the tail biters vs. receivers. In total, 19 genes showed a different expression pattern in neutral pigs when compared with both performers and receivers. This implies that the functions of these may provide knowledge about why the neutral pigs are not involved in tail biting behavior as performers or receivers. Among these 19 transcripts were genes associated with production traits in pigs (PDK4), sociality in humans and mice (GTF2I) and novelty seeking in humans (EGF). These are in line with hypotheses linking tail biting with reduced back fat thickness and explorative behavior.

  • 22. Brunberg, Emma
    et al.
    Jensen, Per
    Isaksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Keeling, Linda J.
    Behavioural and Brain Gene Expression Profiling in Pigs during Tail Biting Outbreaks - Evidence of a Tail Biting Resistant Phenotype2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6, p. e66513-Article in journal (Refereed)
    Abstract [en]

    Abnormal tail biting behaviour is a major welfare problem for pigs receiving the behaviour, as well as an indication of decreased welfare in the pigs performing it. However, not all pigs in a pen perform or receive tail biting behaviour and it has recently been shown that these 'neutral' pigs not only differ in their behaviour, but also in their gene expression compared to performers and receivers of tail biting in the same pen. To investigate whether this difference was linked to the cause or a consequence of them not being involved in the outbreak of tail biting, behaviour and brain gene expression was compared with 'control' pigs housed in pens with no tail biting. It was shown that the pigs housed in control pens performed a wider variety of pig-directed abnormal behaviour (belly nosing 0.95 +/- 1.59, tail in mouth 0.31 +/- 0.60 and 'other' abnormal 1.53 +/- 4.26; mean +/- S.D) compared to the neutral pigs (belly nosing 0.30 +/- 0.62, tail in mouth 0.13 +/- 0.50 and "other" abnormal 0.42 +/- 1.06). With Affymetrix gene expression arrays, 107 transcripts were identified as differently expressed (p < 0.05) between these two categories of pigs. Several of these transcripts had already been shown to be differently expressed in the neutral pigs when they were compared to performers and receivers of tail biting in the same pen in an earlier study. Hence, the different expression of these genes cannot be a consequence of the neutral pigs not being involved in tail biting behaviour, but rather linked to the cause contributing to why they were not involved in tail biting interactions. These neutral pigs seem to have a genetic and behavioural profile that somehow contributes to them being resistant to performing or receiving pig-directed abnormal behaviour, such as tail biting, even when housed in an environment that elicits that behaviour in other pigs.

  • 23.
    Bäcklin, Christofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Machine Learning Based Analysis of DNA Methylation Patterns in Pediatric Acute Leukemia2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer in the Nordic countries. Recent evidence indicate that DNA methylation (DNAm) play a central role in the development and progression of the disease.

    DNAm profiles of a collection of ALL patient samples and a panel of non-leukemic reference samples were analyzed using the Infinium 450k methylation assay. State-of-the-art machine learning algorithms were used to search the large amounts of data produced for patterns predictive of future relapses, in vitro drug resistance, and cytogenetic subtypes, aiming at improving our understanding of the disease and ultimately improving treatment.

    In paper I, the predictive modeling framework developed to perform the analyses of DNAm dataset was presented. It focused on uncompromising statistical rigor and computational efficiency, while allowing a high level of modeling flexibility and usability. In paper II, the DNAm landscape of ALL was comprehensively characterized, discovering widespread aberrant methylation at diagnosis strongly influenced by cytogenetic subtype. The aberrantly methylated regions were enriched for genes repressed by polycomb group proteins, repressively marked histones in healthy cells, and genes associated with embryonic development. A consistent trend of hypermethylation at relapse was also discovered. In paper III, a tool for DNAm-based subtyping was presented, validated using blinded samples and used to re-classify samples with incomplete phenotypic information. Using RNA-sequencing, previously undetected non-canonical aberrations were found in many re-classified samples. In paper IV, the relationship between DNAm and in vitro drug resistance was investigated and predictive signatures were obtained for seven of the eight therapeutic drugs studied. Interpretation was challenging due to poor correlation between DNAm and gene expression, further complicated by the discovery that random subsets of the array can yield comparable classification accuracy. Paper V presents a novel Bayesian method for multivariate density estimation with variable bandwidths. Simulations showed comparable performance to the current state-of-the-art methods and an advantage on skewed distributions.

    In conclusion, the studies characterize the information contained in the aberrant DNAm patterns of ALL and assess its predictive capabilities for future relapses, in vitro drug sensitivity and subtyping. They also present three publicly available tools for the scientific community to use.

    List of papers
    1. Developer Friendly and Computationally Efficient Predictive Modeling without Information Leakage: The emil Package for R
    Open this publication in new window or tab >>Developer Friendly and Computationally Efficient Predictive Modeling without Information Leakage: The emil Package for R
    (English)In: Journal of Statistical Software, ISSN 1548-7660, E-ISSN 1548-7660Article in journal (Other academic) Submitted
    Abstract [en]

    Machine learning-based solutions to predictive modeling problems (classification, regression, or survival analysis) typically involve a number of steps beginning with data pre-processing and ending with performance evaluation. A large number of packages providing tools for the individual steps are available for R but not for facilitating the assembly of them into complete modeling procedures or rigorously evaluating their combined performance.

    We present a new package for R denoted emil (evaluation of modeling without information leakage) that is designed to be a flexible backbone of modeling procedures having the following properties:(1) Enable evaluation of performance and variable importance by means of resampling methods without introducing information leakage.(2) Return parameter tuning statistics and final prediction models.(3) Transparent, highly customizable and easy to debug structure.(4) Offer the user direct control over memory and CPU-intensive steps of the calculations.(5) Comprehensive yet concise documentation.

    First we explain emil's functionality in the context of standard usage, resampling, and customization. Specific application examples are presented to show its potential in terms of parallelization, customization for survival analysis, and memory management.

    The result is a computationally efficient and developer friendly framework that enables resampling based analyzes using several hundreds of thousands of variables, is easy to extend, and allows development of scalable solutions.

    Keywords
    predictive modeling, machine learning, performance evaluation, resampling, high performance computing
    National Category
    Computational Mathematics
    Research subject
    Materials Science
    Identifiers
    urn:nbn:se:uu:diva-242353 (URN)
    Funder
    Swedish Foundation for Strategic Research , RBc08-008
    Available from: 2015-01-25 Created: 2015-01-25 Last updated: 2017-12-05Bibliographically approved
    2. Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia
    Open this publication in new window or tab >>Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia
    Show others...
    2013 (English)In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 14, no 9, p. r105-Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND:

    Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.

    RESULTS:

    We surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.

    CONCLUSIONS:

    Our results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.

    National Category
    Medical Genetics
    Identifiers
    urn:nbn:se:uu:diva-208296 (URN)10.1186/gb-2013-14-9-r105 (DOI)000328195700011 ()24063430 (PubMedID)
    Note

    De två första författarna delar förstaförfattarskapet.

    Available from: 2013-09-27 Created: 2013-09-27 Last updated: 2018-01-11Bibliographically approved
    3. DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia
    Open this publication in new window or tab >>DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia
    Show others...
    2015 (English)In: Clinical Epigenetics, E-ISSN 1868-7083, Vol. 7, article id 11Article in journal (Refereed) Published
    Abstract [en]

    Background

    We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL.

    Results

    We used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (‘other’ subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5.

    Conclusions

    Our findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.

    National Category
    Hematology
    Identifiers
    urn:nbn:se:uu:diva-242351 (URN)10.1186/s13148-014-0039-z (DOI)000350260800001 ()25729447 (PubMedID)
    Funder
    Swedish Foundation for Strategic Research , RBc08-008
    Note

    De två sista författarna delar sistaförfattarskapet.

    Available from: 2015-01-25 Created: 2015-01-25 Last updated: 2017-12-05Bibliographically approved
    4. DNA methylation-based prediction of in vitro drug resistance in primary pediatric acute lymphoblastic leukemia patient samples
    Open this publication in new window or tab >>DNA methylation-based prediction of in vitro drug resistance in primary pediatric acute lymphoblastic leukemia patient samples
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cancer and Oncology Hematology Bioinformatics (Computational Biology)
    Identifiers
    urn:nbn:se:uu:diva-242543 (URN)
    Funder
    Swedish Foundation for Strategic Research , RBc08-008
    Available from: 2015-01-27 Created: 2015-01-27 Last updated: 2018-01-11
    5. Bayesian model averaging of adaptive bandwidth kernel density estimators yields state-of-the-art performance
    Open this publication in new window or tab >>Bayesian model averaging of adaptive bandwidth kernel density estimators yields state-of-the-art performance
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Variable kernel density estimation, adaptive kernel density estimation, Bayesian model averaging, variable bandwidth, square root law
    National Category
    Probability Theory and Statistics
    Identifiers
    urn:nbn:se:uu:diva-242354 (URN)
    Funder
    Swedish Foundation for Strategic Research , RBc08-008EU, FP7, Seventh Framework Programme, PROACTIVE
    Available from: 2015-01-27 Created: 2015-01-25 Last updated: 2015-03-11
  • 24.
    Bäcklin, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bayesian model averaging of adaptive bandwidth kernel density estimators yields state-of-the-art performanceManuscript (preprint) (Other academic)
  • 25.
    Bäcklin, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Self-tuning density estimation based on Bayesian averaging of adaptive kernel density estimations yields state-of-the-art performance2018In: Pattern Recognition, ISSN 0031-3203, E-ISSN 1873-5142, Vol. 78, p. 133-143Article in journal (Refereed)
    Abstract [en]

    Non-parametric probability density function (pdf) estimation is a general problem encountered in many fields. A promising alternative to the dominating solutions, kernel density estimation (KDE) and Gaussian mixture modeling, is adaptive KDE where kernels are given individual bandwidths adjusted to the local data density. Traditionally the bandwidths are selected by a non-linear transformation of a pilot pdf estimate, containing parameters controlling the scaling, but identifying parameters values yielding competitive performance has turned out to be non-trivial. We present a new self-tuning (parameter free) pdf estimation method called adaptive density estimation by Bayesian averaging (ADEBA) that approximates pdf estimates in the form of weighted model averages across all possible parameter values, weighted by their Bayesian posterior calculated from the data. ADEBA is shown to be simple, robust, competitive in comparison to the current practice, and easily generalize to multivariate distributions. An implementation of the method for R is publicly available.

  • 26.
    Bäcklin, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Freyhult, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Frost, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics.
    Palle, Josefine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Lönnerholm, Gudmar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health, Pediatrics.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    DNA methylation-based prediction of in vitro drug resistance in primary pediatric acute lymphoblastic leukemia patient samplesManuscript (preprint) (Other academic)
  • 27.
    Bäcklin, Christofer
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Developer Friendly and Computationally Efficient Predictive Modeling without Information Leakage: The emil Package for RIn: Journal of Statistical Software, ISSN 1548-7660, E-ISSN 1548-7660Article in journal (Other academic)
    Abstract [en]

    Machine learning-based solutions to predictive modeling problems (classification, regression, or survival analysis) typically involve a number of steps beginning with data pre-processing and ending with performance evaluation. A large number of packages providing tools for the individual steps are available for R but not for facilitating the assembly of them into complete modeling procedures or rigorously evaluating their combined performance.

    We present a new package for R denoted emil (evaluation of modeling without information leakage) that is designed to be a flexible backbone of modeling procedures having the following properties:(1) Enable evaluation of performance and variable importance by means of resampling methods without introducing information leakage.(2) Return parameter tuning statistics and final prediction models.(3) Transparent, highly customizable and easy to debug structure.(4) Offer the user direct control over memory and CPU-intensive steps of the calculations.(5) Comprehensive yet concise documentation.

    First we explain emil's functionality in the context of standard usage, resampling, and customization. Specific application examples are presented to show its potential in terms of parallelization, customization for survival analysis, and memory management.

    The result is a computationally efficient and developer friendly framework that enables resampling based analyzes using several hundreds of thousands of variables, is easy to extend, and allows development of scalable solutions.

  • 28.
    Carlier, Charlotte
    et al.
    Univ Ghent, Dept Surg, Expt Surg Lab, Ghent, Belgium..
    Strese, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Viktorsson, Kristina
    Karolinska Inst, Dept Pathol & Oncol, Karolinska Biom Ctr, Stockholm, Sweden..
    Velander, Ebba
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Uustalu, Maria
    Oncopeptides AB, Stockholm, Sweden..
    Juntti, Therese
    Karolinska Inst, Dept Pathol & Oncol, Karolinska Biom Ctr, Stockholm, Sweden.;Oncopeptides AB, Stockholm, Sweden..
    Lewensohn, Rolf
    Karolinska Inst, Dept Pathol & Oncol, Karolinska Biom Ctr, Stockholm, Sweden..
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Spira, Jack
    InSpira Med AB, Tyreso, Sweden..
    De Vlieghere, Elly
    Univ Ghent, Lab Expt Canc Res, Radiat Oncol & Expt Canc Res, Ghent, Belgium..
    Ceelen, Wim P.
    Univ Ghent, Dept Surg, Expt Surg Lab, Ghent, Belgium..
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Preclinical activity of melflufen (J1) in ovarian cancer2016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 37, p. 59322-59335Article in journal (Refereed)
    Abstract [en]

    Ovarian cancer carries a significant mortality. Since symptoms tend to be minimal, the disease is often diagnosed when peritoneal metastases are already present. The standard of care in advanced ovarian cancer consists of platinum-based chemotherapy combined with cytoreductive surgery. Unfortunately, even after optimal cytoreduction and adjuvant chemotherapy, most patients with stage III disease will develop a recurrence. Intraperitoneal administration of chemotherapy is an alternative treatment for patients with localized disease. The pharmacological and physiochemical properties of melflufen, a peptidase potentiated alkylator, raised the hypothesis that this drug could be useful in ovarian cancer and particularily against peritoneal carcinomatosis. In this study the preclinical effects of melflufen were investigated in different ovarian cancer models. Melflufen was active against ovarian cancer cell lines, primary cultures of patient-derived ovarian cancer cells, and inhibited the growth of subcutaneous A2780 ovarian cancer xenografts alone and when combined with gemcitabine or liposomal doxorubicin when administered intravenously. In addition, an intra-and subperitoneal xenograft model showed activity of intraperitoneal administered melflufen for peritoneal carcinomatosis, with minimal side effects and modest systemic exposure. In conclusion, results from this study support further investigations of melflufen for the treatment of peritoneal carcinomatosis from ovarian cancer, both for intravenous and intraperitoneal administration.

  • 29.
    Crona, Joakim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Experimental Surgery.
    Backman, Samuel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Experimental Surgery.
    Maharjan, Rajani
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Experimental Surgery.
    Mayrhofer, Markus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Stålberg, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Isaksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Hellman, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Björklund, Peyman
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Spatiotemporal Heterogeneity Characterizes the Genetic Landscape of Pheochromocytoma and Defines Early Events in Tumorigenesis.2015In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 21, no 19, p. 4451-4460Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Pheochromocytoma and paraganglioma (PPGL) patients display heterogeneity in the clinical presentation and underlying genetic cause. The degree of inter- and intratumor genetic heterogeneity has not yet been defined.

    EXPERIMENTAL DESIGN: In PPGLs from 94 patients, we analyzed LOH, copy-number variations, and mutation status of SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, NF1, RET, TMEM127, MAX, and HRAS using high-density SNP array and targeted deep sequencing, respectively. Genetic heterogeneity was determined through (i) bioinformatics analysis of individual samples that estimated absolute purity and ploidy from SNP array data and (ii) comparison of paired tumor samples that allowed reconstruction of phylogenetic trees.

    RESULTS: Mutations were found in 61% of the tumors and correlated with specific patterns of somatic copy-number aberrations (SCNA) and degree of nontumoral cell admixture. Intratumor genetic heterogeneity was observed in 74 of 136 samples using absolute bioinformatics estimations and in 22 of 24 patients by comparison of paired samples. In addition, a low genetic concordance was observed between paired primary tumors and distant metastases. This allowed for reconstructing the life history of individual tumors, identifying somatic mutations as well as copy-number loss of 3p and 11p (VHL subgroup), 1p (Cluster 2), and 17q (NF1 subgroup) as early events in PPGL tumorigenesis.

    CONCLUSIONS: Genomic landscapes of PPGL are specific to mutation subtype and characterized by genetic heterogeneity both within and between tumor lesions of the same patient.

  • 30.
    Daskalakis, Kosmas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Norlén, Olov
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Karakatsanis, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Hellman, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Stålberg, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Ex vivo activity of cytotoxic drugs and targeted agents in Small Intestinal NETsManuscript (preprint) (Other academic)
    Abstract [en]

    Introduction: Small Intestinal Neuroendocrine Tumours (SI-NET) are considered to be generally resistant to systemic treatment. To date predictive markers for drug activity are lacking.

    Patients and Methods: Tumour samples from 27 patients with SI-NET were analyzed ex vivo for sensitivity to a panel of cytotoxic drugs and targeted agents using a short-term total cell kill assay. Samples of renal cancer, colorectal cancer (CRC), ovarian cancer, and chronic lymphocytic leukemia (CLL) were included for comparison. For the SI-NET subset, drug sensitivity was analyzed in relation to clinico-pathological variables and pre-treatment biomarkers.

    Results: For standard cytotoxic drugs, SI-NETs demonstrated similar or higher sensitivity to 5-FU, platinums, gemcitabine and doxorubicin compared with CRC. For targeted kinase inhibitors, SI-NET was among the most sensitive diagnoses. CLL and ovarian cancer were generally the most sensitive diagnoses to both cytotoxic drugs and protein kinase inhibitors. The mTOR inhibitor sirolimus exhibited modest cytotoxic activity.

    Individual SI-NET samples demonstrated great variability in ex vivo sensitivity for most drugs. Cross-resistance between different drugs also varied considerably, being higher among protein kinase inhibitors.

    Age, stage, grade, peritoneal carcinomatosis and extra-abdominal metastases as well as serum chromogranin A and urine 5-HIAA concentrations at diagnosis did not correlate to drug sensitivity ex vivo.

    Conclusions: SI-NETs exhibit variable but generally intermediate sensitivity ex vivo to cytotoxic and targeted drugs. Clinico-pathological factors and currently used biomarkers were not clearly associated to ex vivo sensitivity, challenging these criteria for treatment decisions in SI-NETs. The great variability in drug sensitivity calls for individualized selection of therapy.

  • 31.
    Daskalakis, Kosmas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Norlén, Olov
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Karakatsanis, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Hellman, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Stålberg, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Ex Vivo Activity of Cytotoxic Drugs and Targeted Agents in Small Intestinal Neuroendocrine Tumors2018In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 106, no Supplement: 1, p. 189-189Article in journal (Other academic)
  • 32.
    Daskalakis, Kosmas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Norlén, Olov
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Karakatsanis, Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Hellman, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Stålberg, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Ex vivo activity of cytotoxic drugs and targeted agents in small intestinal neuroendocrine tumors2018In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 25, no 4, p. 471-480Article in journal (Refereed)
    Abstract [en]

    Small intestinal neuroendocrine tumors (SI-NETs) are generally considered resistant to systemic treatment. To date, predictive markers for drug activity are lacking. Tumor samples from 27 patients with SI-NETs were analyzed ex vivo for sensitivity to a panel of cytotoxic drugs and targeted agents using a short-term total cell kill assay. Samples of renal cancer, colorectal cancer (CRC), ovarian cancer and chronic lymphocytic leukemia (CLL) were included for comparison. For the SI-NET subset, drug sensitivity was analyzed in relation to clinicopathological variables and pre-treatment biomarkers. For cytotoxic drugs, SI-NETs demonstrated similar or higher sensitivity to 5-FU, platinum, gemcitabine and doxorubicin compared with CRC. For several of the targeted kinase inhibitors, SI-NET was among the most sensitive solid tumor types. CLL and ovarian cancer were generally the most sensitive tumor types to both cytotoxic drugs and protein kinase inhibitors. SI-NET was more sensitive to the mTOR inhibitor sirolimus than the other solid tumor types tested. Individual SI-NET samples demonstrated great variability in ex vivo sensitivity for most drugs. Cross-resistance between different drugs also varied considerably, being higher among protein kinase inhibitors. Age, stage, grade, peritoneal carcinomatosis and extra-abdominal metastases as well as serum chromogranin A and urine 5-HIAA concentrations at diagnosis did not correlate to drug sensitivity ex vivo. SI-NETs exhibit intermediate sensitivity ex vivo to cytotoxic and targeted drugs. Clinicopathological factors and currently used biomarkers are not clearly associated to ex vivo sensitivity, challenging these criteria for treatment decisions in SI-NET. The great variability in drug sensitivity calls for individualized selection of therapy.

  • 33.
    De Rosa, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lu, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Zamaratski, Edouard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Szałaj, Natalia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Cao, Sha
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wadensten, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lenhammar, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Roos, Annette K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Huseby, Douglas L
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andrén, Per E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, synthesis and in vitro biological evaluation of oligopeptides targeting E. coli type I signal peptidase (LepB)2017In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 3, p. 897-911Article in journal (Refereed)
    Abstract [en]

    Type I signal peptidases are potential targets for the development of new antibacterial agents. Here we report finding potent inhibitors of E. coli type I signal peptidase (LepB), by optimizing a previously reported hit compound, decanoyl-PTANA-CHO, through modifications at the N- and C-termini. Good improvements of inhibitory potency were obtained, with IC50s in the low nanomolar range. The best inhibitors also showed good antimicrobial activity, with MICs in the low μg/mL range for several bacterial species. The selection of resistant mutants provided strong support for LepB as the target of these compounds. The cytotoxicity and hemolytic profiles of these compounds are not optimal but the finding that minor structural changes cause the large effects on these properties suggests that there is potential for optimization in future studies.

  • 34.
    Delforoush, Maryam
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Strese, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Wickström, Malin
    Univ Uppsala Hosp, Dept Med Sci, Clin Pharmacol Sect, Uppsala, Sweden; Karolinska Inst, Dept Womens & Childrens Hlth, Childhood Canc Res Unit, Stockholm, Sweden.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    In vitro and in vivo activity of melflufen (J1) in lymphoma2016In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 16, article id 263Article in journal (Refereed)
    Abstract [en]

    Background: Melphalan has been used in the treatment of various hematologic malignancies for almost 60 years. Today it is part of standard therapy for multiple myeloma and also as part of myeloablative regimens in association with autologous allogenic stem cell transplantation. Melflufen (melphalan flufenamide ethyl ester, previously called J1) is an optimized derivative of melphalan providing targeted delivery of active metabolites to cells expressing aminopeptidases. The activity of melflufen has compared favorably with that of melphalan in a series of in vitro and in vivo experiments performed preferentially on different solid tumor models and multiple myeloma. Melflufen is currently being evaluated in a clinical phase I/II trial in relapsed or relapsed and refractory multiple myeloma.

    Methods: Cytotoxicity of melflufen was assayed in lymphoma cell lines and in primary tumor cells with the Fluorometric Microculture Cytotoxicity Assay and cell cycle analyses was performed in two of the cell lines. Melflufen was also investigated in a xenograft model with subcutaneous lymphoma cells inoculated in mice.

    Results: Melflufen showed activity with cytotoxic IC50-values in the submicromolar range (0.011-0.92 μM) in the cell lines, corresponding to a mean of 49-fold superiority (p < 0.001) in potency vs. melphalan. In the primary cultures melflufen yielded slightly lower IC50-values (2.7 nM to 0.55 μM) and an increased ratio vs. melphalan (range 13–455, average 108, p < 0.001). Treated cell lines exhibited a clear accumulation in the G2/M-phase of the cell cycle. Melflufen also showed significant activity and no, or minimal side effects in the xenografted animals.

    Conclusion: This study confirms previous reports of a targeting related potency superiority of melflufen compared to that of melphalan. Melflufen was active in cell lines and primary cultures of lymphoma cells, as well as in a xenograft model in mice and appears to be a candidate for further evaluation in the treatment of this group of malignant diseases.

  • 35.
    Delforoush, Maryam
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Sun, Chao
    Thomas, Strömberg
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Strese, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Inhibition of the 19 S proteasome by bAP-15 in lymphoma cell linesManuscript (preprint) (Other academic)
    Abstract [en]

    Inhibition of activity of proteasome by bortezomib has been shown to selectively kill cancer cells. Bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma and the proteolytic 20S core particle of the proteasome, has also been clinically validated as a therapeutic target in oncology. However, despite its acceptable therapeutic index, patients treated with bortezomib show toxic side effects and eventually acquire resistance to the drug. A lot of efforts are currently been made to develop new proteasome inhibitors that perform through mechanisms different from that of bortezomib.

    We have studied the effects of b-AP15, a novel inhibitor of the deubiquitinase activity in the 19S regulatory subunit of the proteasome, on a panel of nine cell-lines from diffuse large B-cell lymphomas (DLBCL) and three from Hodgkin lymphoma (HL). All cell lines showed a dose dependent reduction of viability. The inhibition of the 19S subunit by b-AP15 resulted, as expected, in accumulation of ubiquitinylated proteins at concentrations close to cytotoxic IC50. Increases in polyubiquitinated proteins were paralleled by increases in the inducible form of heat shock protein 70 (Hsp70B´), and a strong association between Hsp70B´ induction and cleavage of PARP and caspase-3 was observed. These data suggest that proteotoxic stress mediates the sensitivity of lymphoma cells to the deubiquitinase inhibitor b-AP15.

    The findings in this study suggest that b-AP15 should further evaluated as a drug in lymphoma treatment.

  • 36.
    Dimberg, Lina Y.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Ivarsson, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Tobin, Gerard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Ekman, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullberg, Urban
    Nilsson, Kenneth
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Öberg, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Wiklund, Helena Jernberg
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Hematology and Immunology.
    Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma2012In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 12, p. 318-Article in journal (Refereed)
    Abstract [en]

    Background: Multiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM. Methods: To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS). Results: To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered. Conclusion: We conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro-apoptotic genes. However, this shift alone is not sufficient to alter apoptosis sensitivity in MM cells, suggesting that Stat1 independent pathways are operative in IFN mediated apoptosis sensitization.

  • 37.
    Edfeldt, Katarina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Daskalakis, Kosmas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Bäcklin, Christofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Norlén, Olov
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Tiensuu Janson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrin Oncology.
    Westin, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Hellman, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Stålberg, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    DcR3, TFF3 and Midkine are Novel Serum Biomarkers in Small Intestinal Neuroendocrine Tumors2017In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 105, no 2, p. 170-181Article in journal (Refereed)
    Abstract [en]

    Small intestinal neuroendocrine tumors (SI-NETs) are amine- and peptide producing neoplasms. Most patients display metastases at the time of diagnosis, they have an unpredictable individual disease course and the tumors are often therapy resistant. Chromogranin A (CgA) and 5-hydroxyindoleacetic acid (5-HIAA) are the clinically most used biomarkers today, but there is a great need for novel diagnostic and prognostic biomarkers and new therapeutic targets. Sixty-nine biomarkers were screened in serum from 23 SI-NET patients and 23 healthy controls using multiplex PLA (proximity ligation assay). A refined method, PEA (proximity extension assay), was used to analyze 76 additional biomarkers. Statistical testing and multivariate classification were performed. Immunohistochemistry and ELISA assays were performed in an extended cohort. Using PLA, 19 biomarkers showed a significant difference in serum concentrations between patients and controls, and PEA revealed difference in concentrations in 13 proteins. Multivariate classification analysis revealed decoy receptor 3 (DcR3), trefoil factor 3 (TFF3) and Midkine to be good biomarkers for disease, which was confirmed by ELISA analysis. All three biomarkers were expressed in tumor tissue. DcR3 concentrations were elevated in patients with stage IV disease. High concentrations of DcR3 and TFF3 were correlated to poor survival. DcR3, TFF3 and Midkine exhibited elevated serum concentrations in SI-NET patients compared to healthy controls, and DcR3 and TFF3 were associated with poor survival. DcR3 seems to be a marker for liver metastases while TFF3 and Midkine may be new diagnostic biomarkers for SI-NETs.

  • 38.
    El-Seedi, Hesham R.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Burman, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Mansour, Ahmed
    Turki, Zaki
    Boulos, Loutfy
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    The traditional medical uses and cytotoxic activities of sixty-one Egyptian plants: Discovery of an active cardiac glycoside from Urginea maritima2013In: Journal of Ethnopharmacology, ISSN 0378-8741, E-ISSN 1872-7573, Vol. 145, no 3, p. 746-757Article in journal (Refereed)
    Abstract [en]

    Ethnopharmacological relevance: Medicinal plants from the Sinai desert are widely used in traditional Bedouin medicine to treat a range of conditions including, cancers, and may thus be useful sources of novel anti-tumor compounds. Information on plants used in this way was obtained through collaboration with Bedouin herbalists. Aim of the study: To document the traditional uses of 61 species from 29 families of Egyptian medicinal plants and to investigate their biological activity using a cytotoxicity assay. Material and methods: MeOH extracts of the 61 plant species investigated were dissolved in 10% DMSO and their cytotoxic activity was evaluated. The extracts were tested in duplicate on three separate occasions at three different concentrations (1, 10 and 100 mu g/ml) against human lymphoma U-937 GTB. The most active extract was subjected to bioassay-guided fractionation using HPLC and LC/ESI-MS to isolate and identify its active components. Results and discussion: The most potent extracts were those from Asclepias sinaica, Urginea maritima, Nerium oleander and Catharanthus roseus, followed by those from Cichorium endivia, Pulicaria undulate and Melia azedarach. Literature reports indicate that several of these plants produce cardiac glycosides. Bioassay-guided fractionation of alcoholic U. maritima extracts led to the isolation of a bioactive bufadienolide that was subsequently shown to be proscillaridin A, as determined by 1D and 2D NMR spectroscopy. This result demonstrates the value of plants used in traditional medicine as sources of medicinally interesting cytotoxic compounds.

  • 39.
    Emami Khoonsari, Payam
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Haggmark, Anna
    KTH Royal Inst Technol, Sch Biotechnol, Affin Prote, Sci Life Lab, Stockholm, Sweden..
    Lönnberg, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Mikus, Maria
    KTH Royal Inst Technol, Sch Biotechnol, Affin Prote, Sci Life Lab, Stockholm, Sweden..
    Kilander, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Nilsson, Peter
    KTH Royal Inst Technol, Sch Biotechnol, Affin Prote, Sci Life Lab, Stockholm, Sweden..
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Shevchenko, Ganna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala Univ, Dept Chem BMC, Analyt Chem, Uppsala, Sweden..
    Analysis of the Cerebrospinal Fluid Proteome in Alzheimer's Disease2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, article id e0150672Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer's disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer's disease patients and non-demented controls to identify potential biomarkers for Alzheimer's disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer's disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer's disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system.

  • 40.
    Eriksson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Studies of New Signal Transduction Modulators in Acute Myeloid Leukemia2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Acute myeloid leukemia (AML) is a life-threatening malignant disorder with dismal prognosis. AML is characterized by frequent genetic changes involving tyrosine kinases, normally acting as important mediators in many basic cellular processes. Due to the overexpression and frequent mutations of the FMS-like receptor tyrosine kinase 3 (FLT3) in AML, this tyrosine kinase receptor has become one of the most sought after targets in AML drug development.

    In this thesis, we have used a combination of high-throughput screens, direct target interaction assays and sequential cellular screens, including primary patient samples, as an approach to discover new targeted therapies. Gefitinib, a previously known inhibitor of epidermal growth factor receptor and the two novel tyrosine kinase inhibitors AKN-032 and AKN-028, have been identified as compounds with cytotoxic activity in AML.

    AKN-028 is a potent inhibitor of FLT3 with an IC50 value of 6 nM in an enzyme assay, but also displaying in vitro activity in a variety of primary AML samples, irrespective of FLT3 mutation status or quantitative FLT3 expression. AKN-028 shows a sequence dependent in vitro synergy when combined with standard cytotoxic agents cytarabine or daunorubicin, with better efficacy when cells are exposed to standard chemotherapy simultaneously or for 24 hours prior to adding AKN-028. Antagonism is observed when cells are pre-treated with AKN-028, possibly explained by the cell cycle arrest induced by the compound. In vivo cytotoxic activity and good oral bioavailability have made AKN-028 a candidate drug for clinical studies and the compound is presently investigated in an international two-part multicenter phase I/II study.

    Results from microarray studies performed to further elucidate the mechanism of action of AKN-028, revealed significantly altered gene expression induced by AKN-028 in both AML cell lines and in primary AML cells, with an enrichment of the Myc pathway among the downregulated genes.

    Furthermore, tyrosine kinase activity profiling shows a dose-dependent kinase inhibition by AKN-028 in all AML samples tested. Interestingly, cells with a high overall kinase activity were more sensitive to AKN-028. Provided conformation in a larger set of samples, kinase activity profiling may give useful information in individualizing treatment of patients with AML.

    List of papers
    1. Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia
    Open this publication in new window or tab >>Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia
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    2008 (English)In: European Journal of Haematology, ISSN 0902-4441, E-ISSN 1600-0609, Vol. 81, no 5, p. 344-353Article in journal (Refereed) Published
    Abstract [en]

    Objectives: 

    Gefitinib inhibits epidermal growth factor receptor (EGFR) signalling, but may also act by non-EGFR dependent mechanisms. We have investigated the activity of gefitinib in haematological tumour cells, in particular acute myeloblastic leukaemia (AML).

    Methods: 

    Cytotoxic activity of gefitinib, alone or in combination with standard anti-leukaemic drugs, was assessed by the short-term fluorometric microculture cytotoxicity assay in tumour cells from 117 patients representing five haematological and five non-haematological malignancies. In AML, the EGFR status was analysed by immunochemistry. Gefitinib-induced apoptosis was investigated in a subset of AML samples, as well as in the leukaemia cell line MV-4-11, using a multiparametric high content screening assay. To confirm activation of caspase-3 in cells treated with gefitinib, a blocking test was carried out in which MV4-11 cells were pretreated with the specific caspase inhibitor DEVD-FMK.

    Results: 

    Gefitinib showed highest cytotoxic activity in AML (= 19) with many samples being sensitive at concentrations achievable in clinical practice (<10 μM), and no difference between previously untreated and relapsed patients. No correlation between the activity of gefitinib and standard antileukaemic drugs (cytarabine, doxorubicin, etoposide) was observed. Combining gefitinib with these drugs resulted in mainly additive or synergistic (etoposide) effects, with no evidence of sequence dependency. The AML cells did not express the EGFR. Gefitinib induced apoptosis, which was at least partly mediated by activation of the caspase-3 pathway.

    Conclusion: 

    In vitro, gefitinib has significant cytotoxic activity in AML by inducing apoptosis through non-EGFR dependent pathways.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-102372 (URN)10.1111/j.1600-0609.2008.01120.x (DOI)000260185700002 ()18637032 (PubMedID)
    Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2017-12-13Bibliographically approved
    2. Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia
    Open this publication in new window or tab >>Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia
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    2010 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 10, p. 1507-1516Article in journal (Refereed) Published
    Abstract [en]

    Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC50 of 70 nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC50 = 0.4 mu M) and Kasumi-1 (IC50 = 2.3 mu M). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion. AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

    Keywords
    Acute myeloid leukemia, New drug development, Tyrosine kinase inhibitor, FLT3
    National Category
    Medical and Health Sciences Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-134176 (URN)10.1016/j.bcp.2010.08.002 (DOI)000282850900006 ()
    Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2018-01-12Bibliographically approved
    3. The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia
    Open this publication in new window or tab >>The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia
    Show others...
    2012 (English)In: Blood Cancer Journal, ISSN 2044-5385, E-ISSN 2044-5385, Vol. 2, p. e81-Article in journal (Refereed) Published
    Abstract [en]

    Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC50=6 nM), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC50 1 μM). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).

    National Category
    Medical and Health Sciences Hematology Pharmacology and Toxicology
    Identifiers
    urn:nbn:se:uu:diva-182062 (URN)10.1038/bcj.2012.28 (DOI)000308664500001 ()22864397 (PubMedID)
    Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2018-01-12Bibliographically approved
    4. AKN-028 induces cell cycle arrest, downregulation of Myc associated genes and a dose dependent reduction of kinase activity in acute myeloid leukemia
    Open this publication in new window or tab >>AKN-028 induces cell cycle arrest, downregulation of Myc associated genes and a dose dependent reduction of kinase activity in acute myeloid leukemia
    Show others...
    2014 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 87, no 2, p. 284-291Article in journal (Refereed) Published
    Abstract [en]

    AKN-028 is a novel tyrosine kinase inhibitor with preclinical activity in acute myeloid leukemia (AML), presently undergoing investigation in a phase I/II study. It is a potent inhibitor of the FMS-like kinase 3 (FLT3) but shows in vitro activity in a wide range of AML samples. In the present study, we have characterized the effects of AKN-028 on AML cells in more detail. AKN-028 induced a dose-dependent G(0)/arrest in AML cell line MV4-11. Treatment with AKN-028 caused significantly altered gene expression in all AML cell types tested (430 downregulated, 280 upregulated transcripts). Subsequent gene set enrichment analysis revealed enrichment of genes associated with the proto-oncogene and cell cycle regulator c-Myc among the downregulated genes in both AKN-028 and midostaurin treated cells. Kinase activity profiling in AML cell lines and primary AML samples showed that tyrosine kinase activity, but not serine/threonine kinase activity, was inhibited by AKN-028 in a dose dependent manner in all samples tested, reaching approximately the same level of kinase activity. Cells sensitive to AKN-028 showed a higher overall tyrosine kinase activity than more resistant ones, whereas serine/threonine kinase activity was similar for all primary AML samples. In summary, AKN-028 induces cell cycle arrest in AML cells, downregulates Myc-associated genes and affect several signaling pathways. AML cells with high global tyrosine kinase activity seem to be more sensitive to the cytotoxic effect of AKN-028 in vitro.

    Place, publisher, year, edition, pages
    Elsevier, 2014
    Keywords
    Acute myeloid leukemia, AKN-028, Tyrosine kinase inhibitor, Signal transduction
    National Category
    Hematology Pharmacology and Toxicology Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-182065 (URN)10.1016/j.bcp.2013.10.022 (DOI)000330332800006 ()
    Available from: 2012-10-10 Created: 2012-10-03 Last updated: 2018-01-12Bibliographically approved
  • 41.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Chantzi, Efthymia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Gustafsson, Mats G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Towards repositioning of quinacrine for treatment of acute myeloid leukemia - Promising synergies and in vivo effects.2017In: Leukemia research: a Forum for Studies on Leukemia and Normal Hemopoiesis, ISSN 0145-2126, E-ISSN 1873-5835, Vol. 63, p. 41-46Article in journal (Refereed)
    Abstract [en]

    We previously reported that the anti-malarial drug quinacrine has potential to be repositioned for treatment of acute myeloid leukemia (AML). As a next step towards clinical use, we assessed the efficacy of quinacrine in an AML-PS mouse model and investigated possible synergistic effects when combining quinacrine with nine other antileukemic compounds in two AML cell lines. Furthermore, we explored the in vivo activity of quinacrine in combination with the widely used AML agent cytarabine. The in vivo use of quinacrine (100mg/kg three times per week for two consecutive weeks) significantly suppressed circulating blast cells at days 30/31 and increased the median survival time (MST). The in vitro drug combination analysis yielded promising synergistic interactions when combining quinacrine with cytarabine, azacitidine and geldanamycin. Finally, combining quinacrine with cytarabine in vivo showed a significant decrease in circulating leukemic blast cells and increased MST compared to the effect of either drug used alone, thus supporting the findings from the in vitro combination experiments. Taken together, the repositioning potential of quinacrine for treatment of AML is reinforced by demonstrating significant in vivo activity and promising synergies when quinacrine is combined with different agents, including cytarabine, the hypomethylating agent azacitidine and HSP-90 inhibitor geldanamycin.

  • 42.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Repositioning Of Quinacrine For Treatment Of Acute Myeloid Leukemia - Synergies And In Vivo Effects2016In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 367-368Article in journal (Other academic)
  • 43.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Hermanson, Monica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics.
    Wickström, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Lindhagen, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Ekholm, C
    Jenmalm Jensen, A
    Löthgren, A
    Lehmann, F
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Parrow, V
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia2012In: Blood Cancer Journal, ISSN 2044-5385, E-ISSN 2044-5385, Vol. 2, p. e81-Article in journal (Refereed)
    Abstract [en]

    Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC50=6 nM), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC50 1 μM). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).

  • 44.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Osterros, Albin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hassan, Sadia Bashir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Repositioning of Quinacrine for Treatment of Acute Myeloid Leukemia2014In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 124, no 21Article in journal (Other academic)
  • 45.
    Eriksson, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Österroos, Albin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hassan, Sadia Bashir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Höglund, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Drug screen in patient cells suggests quinacrine to be repositioned for treatment of acute myeloid leukemia2015In: Blood Cancer Journal, ISSN 2044-5385, E-ISSN 2044-5385, Vol. 5, article id e307Article in journal (Refereed)
    Abstract [en]

    To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC1280 library (Sigma). Twenty-five compounds were defined as hits with activity in all leukemia subgroups (<50% cell survival compared with control) at 10 mu M drug concentration. Only one of these compounds, quinacrine, showed low activity in normal PBMCs and was therefore selected for further preclinical evaluation. Mining the NCI-60 and the NextBio databases demonstrated leukemia sensitivity and the ability of quinacrine to reverse myeloid leukemia gene expression. Mechanistic exploration was performed using the NextBio bioinformatic software using gene expression analysis of drug exposed acute myeloid leukemia cultures (HL-60) in the database. Analysis of gene enrichment and drug correlations revealed strong connections to ribosomal biogenesis nucleoli and translation initiation. The highest drug-drug correlation was to ellipticine, a known RNA polymerase I inhibitor. These results were validated by additional gene expression analysis performed in-house. Quinacrine induced early inhibition of protein synthesis supporting these predictions. The results suggest that quinacrine have repositioning potential for treatment of acute myeloid leukemia by targeting of ribosomal biogenesis.

  • 46. Fawzy, Iten M.
    et al.
    Youssef, Khairia M.
    Ismail, Nasser S. M.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Abouzid, Khaled A. M.
    Newly Designed and Synthesized Curcumin Analogs with in vitro Cytotoxicity and Tubulin Polymerization Activity2015In: Chemical Biology and Drug Design, ISSN 1747-0277, E-ISSN 1747-0285, Vol. 86, no 1, p. 860-870Article in journal (Refereed)
    Abstract [en]

    Novel curcumin analogs with 4-piperidone ring were designed, synthesized, and evaluated for their cytotoxic activities against five different cancer cell lines. 3,5-bis(4-Hydroxy-3-methoxybenzylidene)-4-oxo-N-phenylpiperidine-1-carbothioamide (XIIe) exhibited considerable cytotoxic activity with IC50 values in 1-2.5m range. In silico and in vitro, studies were also performed to predict the binding affinity of the target compounds to the -chain of tubulin receptor (PDB code 1SA1) and their abilities to affect microtubules polymerization cycle. 3,5-bis(3-Iodo-5-methoxy-4-propoxybenzylidene)-N-acetylpiperidin-4-one (VIIa) was found to exert 93.3% inhibition of tubulin and destabilization of microtubules in vitro compared to vincristine while, 3,5-bis(3,4,5-trimethoxybenzylidene)-N-benzoylpiperidin-4-one (XIIc) showed high potency in a differentway where it exerted 94.8% stabilization of microtubules in vitro compared to positive control paclitaxel.

  • 47.
    Felth, Jenny
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Lesiak-Mieczkowska, Karolina
    Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute.
    Haglund, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Linder, Stig
    Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute.
    Bohlin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gambogic acid is cytotoxic to cancer cells through inhibition of the ubiquitin-proteasome system2013In: Investigational new drugs, ISSN 0167-6997, E-ISSN 1573-0646, Vol. 31, no 3, p. 587-598Article in journal (Other academic)
    Abstract [en]

    Gambogic acid (GA), displays cytotoxicity towards a wide variety of tumor cells and has been shown to affect many important cell-signaling pathways. In the present work, we investigated the mechanism of action of GA by analysis of drug-induced changes in gene expression profiles and identified GA and the derivative dihydro GA as possible inhibitors of the ubiquitin-proteasome system (UPS). Both GA and dihydro GA inhibited proteasome function in cells resulting in the accumulation of polyubiquitin complexes. In vitro experiments showed that both GA and dihydro GA inhibited 20S chymotrypsin activity and the inhibitory effects of GA and dihydro GA on proteasome function corresponded with apoptosis induction and cell death. In conclusion, our results show that GA and dihydro GA exert their cytotoxic activity through inhibition of the UPS, specifically by acting as inhibitors of the chymotrypsin activity of the 20S proteasome.

  • 48.
    Freyhult, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gustafsson, Mats G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Strömbergsson, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    A Machine Learning Approach to Explain Drug Selectivity to Soluble and Membrane Protein Targets2015In: Molecular Informatics, ISSN 1868-1751, Vol. 34, no 1, p. 44-52Article in journal (Refereed)
  • 49.
    Fryknäs, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Wang, Xin
    Rickardson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Wickström, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Hassan, Saadia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Westman, Gunnar
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Oncology.
    Linder, Stig
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Screening for phenotype selective activity in multidrug resistant cells identifies a novel tubulin active agent insensitive to common forms of cancer drug resistance2013In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 13, p. 374-Article in journal (Refereed)
    Abstract [en]

    Background: Drug resistance is a common cause of treatment failure in cancer patients and encompasses a multitude of different mechanisms. The aim of the present study was to identify drugs effective on multidrug resistant cells. Methods: The RPMI 8226 myeloma cell line and its multidrug resistant subline 8226/Dox40 was screened for cytotoxicity in response to 3,000 chemically diverse compounds using a fluorometric cytotoxicity assay (FMCA). Follow-up profiling was subsequently performed using various cellular and biochemical assays. Results: One compound, designated VLX40, demonstrated a higher activity against 8226/Dox40 cells compared to its parental counterpart. VLX40 induced delayed cell death with apoptotic features. Mechanistic exploration was performed using gene expression analysis of drug exposed tumor cells to generate a drug-specific signature. Strong connections to tubulin inhibitors and microtubule cytoskeleton were retrieved. The mechanistic hypothesis of VLX40 acting as a tubulin inhibitor was confirmed by direct measurements of interaction with tubulin polymerization using a biochemical assay and supported by demonstration of G2/M cell cycle arrest. When tested against a broad panel of primary cultures of patient tumor cells (PCPTC) representing different forms of leukemia and solid tumors, VLX40 displayed high activity against both myeloid and lymphoid leukemias in contrast to the reference compound vincristine to which myeloid blast cells are often insensitive. Significant in vivo activity was confirmed in myeloid U-937 cells implanted subcutaneously in mice using the hollow fiber model. Conclusions: The results indicate that VLX40 may be a useful prototype for development of novel tubulin active agents that are insensitive to common mechanisms of cancer drug resistance.

  • 50.
    Fryknäs, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Zhang, Xiaonan
    Linkping Univ, Dept Med & Hlth Sci, SE-58183 Linkoping, Sweden.;Karolinska Inst, Dept Pathol & Oncol, Canc Ctr Karolinska, SE-17176 Stockholm, Sweden..
    Bremberg, Ulf
    Beactica, SE-75450 Uppsala, Sweden..
    Senkowski, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Olofsson, Maria Hägg
    Karolinska Inst, Dept Pathol & Oncol, Canc Ctr Karolinska, SE-17176 Stockholm, Sweden..
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Persson, Ingmar
    Swedish Univ Agr Sci, Dept Chem & Biotechnol, POB 7015, SE-75651 Uppsala, Sweden..
    D'Arcy, Padraig
    Linkping Univ, Dept Med & Hlth Sci, SE-58183 Linkoping, Sweden..
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Schughart, Leoni Kunz
    Tech Univ Dresden, OncoRay Natl Ctr Radiat Res Oncol, D-01307 Dresden, Germany..
    Linder, Stig
    Linkping Univ, Dept Med & Hlth Sci, SE-58183 Linkoping, Sweden.;Karolinska Inst, Dept Pathol & Oncol, Canc Ctr Karolinska, SE-17176 Stockholm, Sweden..
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Iron chelators target both proliferating and quiescent cancer cells2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38343Article in journal (Refereed)
    Abstract [en]

    Poorly vascularized areas of solid tumors contain quiescent cell populations that are resistant to cell cycle-active cancer drugs. The compound VLX600 was recently identified to target quiescent tumor cells and to inhibit mitochondrial respiration. We here performed gene expression analysis in order to characterize the cellular response to VLX600. The compound-specific signature of VLX600 revealed a striking similarity to signatures generated by compounds known to chelate iron. Validation experiments including addition of ferrous and ferric iron in excess, EXAFS measurements, and structure activity relationship analyses showed that VLX600 chelates iron and supported the hypothesis that the biological effects of this compound is due to iron chelation. Compounds that chelate iron possess anti-cancer activity, an effect largely attributed to inhibition of ribonucleotide reductase in proliferating cells. Here we show that iron chelators decrease mitochondrial energy production, an effect poorly tolerated by metabolically stressed tumor cells. These pleiotropic features make iron chelators an attractive option for the treatment of solid tumors containing heterogeneous populations of proliferating and quiescent cells.

1234 1 - 50 of 157
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