Inference of kinase-signaling networks in human myeloid cell line models by Phosphoproteomics using kinase activity enrichment analysis (KAEA)

Background Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance. Methods We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4–11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors. Results Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4–11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4–11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. Conclusions We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples. Graphical abstract

Changes in Intestinal Permeability Ex Vivo and Immune Cell Activation by Three Commonly Used Emulsifiers

Food additives such as emulsifiers are used in increasing quantities in the food industry. The aim of this study was to compare three different emulsifiers (polysorbate 80 (P80), carboxymethyl cellulose (CMC), and β-lactoglobulin (β-lac) with regards to their effect on the stimulation of immune cells and intestinal permeability. The immune stimulatory effects were studied in the myeloid cell line MUTZ-3-cells, while the change in intestinal permeability was studied in the Caco-2 cell line and ex vivo in the Ussing chamber system using small intestinal fragments from rats. The tested concentrations of the emulsifiers ranged from 0.02% up to 1%, which are concentrations commonly used in the food industry. The results showed that P80 affected both the myeloid cells and the intestinal permeability more than CMC (p < 0.05) and β-lac (p < 0.05) at the highest concentration. CMC was found to neither affect the permeability in the intestine nor the MUTZ-3 cells, while β-lac changed the permeability in the total part of the small intestine in rats. These findings indicate that P80 might be more cytotoxic compared to the other two emulsifiers.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

Background The U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements. Results SNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances. Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified. There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss. Conclusions Centromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

The Potentials and Pitfalls of a Human Cervical Organoid Model Including Langerhans Cells

Three-dimensional cell culturing to capture a life-like experimental environment has become a versatile tool for basic and clinical research. Mucosal and skin tissues can be grown as “organoids” in a petri dish and serve a wide variety of research questions. Here, we report our experience with human cervical organoids which could also include an immune component, e.g., Langerhans cells. We employ commercially available human cervical keratinocytes and fibroblasts as well as a myeloid cell line matured and purified into langerin-positive Langerhans cells. These are then seeded on a layer of keratinocytes with underlying dermal equivalent. Using about 10-fold more than the reported number in healthy cervical tissue (1–3%), we obtain differentiated cervical epithelium after 14 days with ~1% being Langerhans cells. We provide a detailed protocol for interested researchers to apply the described “aseptic” organoid model for all sorts of investigations—with or without Langerhans cells.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

BackgroundThe U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements. ResultsSNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances.Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified.There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss.Conclusions Centromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

SOD2 ameliorates pulmonary hypertension in a murine model of sleep apnea via suppressing expression of NLRP3 in CD11b+ cells

Background High prevalence of obstructive sleep apnea (OSA) in the pulmonary hypertension (PH) population suggests that chronic intermittent hypoxia (CIH) is an important pathogenic factor of PH. However, the exact mechanism of CIH induced PH is not clear. One of the molecules that plays a key role in regulating pulmonary artery function under hypoxic conditions is superoxide dismutase 2 (SOD2). Methods Our study utilized heterozygous SOD2−/+ mice firstly in CIH model to explore the exact role of SOD2 in CIH causing PH. Expression of SOD2 was analyzed in CIH model. Echocardiography and pulmonary hypertension were measured in wild type (WT) and SOD2−/+ mice under normal air or CIH condition. Hematoxylin–Eosin (H&E) staining and masson staining were carried out to evaluate pulmonary vascular muscularization and remodeling. Micro-PET scanning of in vivo 99mTc-labelled- MAG3-anti-CD11b was applied to assess CD11b in quantification and localization. Level of nod-like receptor pyrin domain containing 3 (NLRP3) was analyzed by real time PCR and immunohistochemistry (IHC). Results Results showed that SOD2 was down-regulated in OSA/CIH model. Deficiency of SOD2 aggravated CIH induced pulmonary hypertension and pulmonary vascular hypertrophy. CD11b+ cells, especially monocytic myeloid cell line-Ly6C+Ly6G− cells, were increased in the lung, bone marrow and the blood under CIH condition, and down-regulated SOD2 activated NLRP3 in CD11b+ cells. SOD2-deficient-CD11b+ myeloid cells promoted the apoptosis resistance and over-proliferation of human pulmonary artery smooth muscle cells (PASMCs) via up-regulating NLRP3. Conclusion CIH induced down-regulating of SOD2 increased pulmonary hypertension and vascular muscularization. It could be one of the mechanism of CIH leading to PH.

Characterization of Bypassing Kinases Inferred By Phosphoproteomics in a Midostaurin Resistant Myeloid Cell Line Model

Background: Chronic myeloid neoplasms are heterogeneous malignancies caused by sequential accumulation of genetic lesions in hematopoietic stem cells with a tendency to evolve towards acute myeloid leukemia. Genomic approaches can stratify patients according to their mutational landscape but are limited in predicting response to therapeutic agents. Reliable biomarkers to identify patients' chances for response to targeting compounds remain a crucial necessity in the current post-genomic era of precision medicine. We hypothesize that differential phosphoproteomic (PP) profiles might represent a suitable functional biological layer that allows to identify relevant determinants of oncogenic phenotypes. Aim: The aim of the project was to build a bioinformatics pipeline using PP data to i) identify differentially phosphorylated sites, ii) infer targetable kinases and iii) characterize involved oncogenic pathways. Here, we present results from further exploratory experiments of a previously established PP analysis pipeline that enables to infer sensitive/bypassing kinase activities in a Midostaurin/PKC412 (MIDO) resistant myeloid cell line model. Methods: For the validation of our analysis pipeline, we have previously used the human myeloid cell lines K562 and MOLM13, driven by the oncogenic BCR-ABL1 and FLT3 kinases, and exposed to the kinase inhibitors Nilotinib (NILO) and MIDO, respectively. For the current biological study, we applied our pipeline to explore MIDO resistance mechanisms in published MIDO-resistant (rMOLM13) and sensitive (sMOLM13) cell lines (kindly provided by E. Weisberg, Dana Farber Cancer Institute, USA). r/sMOLM13 were cultured in triplicates for 24hrs and subsequently exposed for 1h to 25nM MIDO or DMSO (CTRL). PPs were enriched with titanium-dioxide and analyzed by mass spectrometry (nanoLC-MS2). Our previously validated Kinase Activity Enrichment Analysis (KAEA) bioinformatics pipeline was further applied to infer kinase activities based on the SetRank enrichment algorithm. KAEA integrates substrate-kinase datasets from five experimentally validated databases complemented with NetworKIN in-silico predictions. The pipeline is supported by a Shiny web-app interface to allow interactive visualization and interrogation of the data. Results: In our previous validation experiments, K562 treated with NILO showed expected inhibition of ABL1 and KIT, whereas MOLM13 treated with MIDO showed expected inhibition of PRKC and downstream kinases of FLT3 (AKT1 and MAPK). In the current biological exploration, we compared rMOLM13 vs sMOLM13 exposed to MIDO and CTRL. rMOLM13 showed slower proliferation and vacuolization compared to sMOLM13. 13,682 phosphorylation sites were identified and the three replicates clustered adequately in the corresponding four conditions sMOLM13_CTRL, sMOLM13_MIDO, rMOLM13_CTRL and rMOLM13_MIDO (fig. 1A). Clusters of over- or underexpressed sites associated with distinct conditions emerged in the heatmap. We performed a KAEA comparison of rMOLM13_MIDO vs sMOLM13_MIDO (fig. 1B) and rMOLM13_CTRL vs sMOLM13_CTRL (fig. 1C). CDK1 and other CDKs enriched as underactive in rMOLM13, in line with the observed reduced proliferation. Most interestingly, kinases PDHK1, PDHK2 and PKM, all pyruvate kinases involved in regulation of mitochondrial metabolism, enriched as overactive, as well as MAPK1/3 and CK2. Reduced proliferation, vacuolization and patterns of enriched kinases pointed towards autophagy as potential resistance mechanism in rMOLM13. We performed preliminary experiments with autophagy inhibitors in combination with MIDO, which supported a synergistic activity (fig. 1D). More results of combined inhibition of the identified kinases will be shown at the meeting. Conclusions: With our PP analysis pipeline, we were able to characterize, in unanticipated detail, dynamic changes of PP profiles, kinase activities and potentially involved oncogenic pathways in a MIDO resistant myeloid cell line model. We identified biologically relevant mechanisms of resistance that will be explored as potential targets for combination therapy. Our experiments in myeloid cell lines provide a proof of concept for our analysis pipeline. They allow us to move forward to the analysis of primary patient samples and promote precision medicine based on functional biomarkers identified by PP in patients with myeloid neoplasms. Disclosures Jankovic: Celgene: Other: financial support for travel. Bonadies:Roche: Other: financial support for travel, Research Funding; Novartis: Other: financial support for travel, Research Funding; Celgene: Other: financial support for travel, Research Funding; Janssen: Other: financial support for travel; Amgen: Other: financial support for travel; Sanofi Genzyme: Other: financial support for travel.

The potentials and pitfalls of a human cervical organoid model including Langerhans cells

Three-dimensional cell culturing to capture a life-like experimental environment has become a versatile tool for basic and clinical research. Mucosal and skin tissues can be grown as “organoids” in a petri dish and serve a wide variety of research questions. Here, we report our experience with human cervical organoids which could also include an immune component, e.g. Langerhans cells. We employ commercially available human cervical keratinocytes and fibroblasts as well as a myeloid cell line matured and purified into langerin-positive Langerhans cells. These are then seeded on a layer of keratinocytes with underlying dermal equivalent. Using about 10-fold more than the reported number in healthy cervical tissue (1–3%), we obtain differentiated cervical epithelium after 14 days with ~ 1% being Langerhans cells. We provide a detailed protocol for interested researchers to apply the described “aseptic” organoid model for all sorts of investigations—with or without Langerhans cells.