Publications by authors named "Annkristin Heine"

37 Publications

Clinical and immunological effects of mRNA vaccines in malignant diseases.

Mol Cancer 2021 03 15;20(1):52. Epub 2021 Mar 15.

Medical Clinic III for Oncology, Hematology, Immune-Oncology and Rheumatology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany.

In vitro-transcribed messenger RNA-based therapeutics represent a relatively novel and highly efficient class of drugs. Several recently published studies emphasize the potential efficacy of mRNA vaccines in treating different types of malignant and infectious diseases where conventional vaccine strategies and platforms fail to elicit protective immune responses. mRNA vaccines have lately raised high interest as potent vaccines against SARS-CoV2. Direct application of mRNA or its electroporation into dendritic cells was shown to induce polyclonal CD4+ and CD8+ mediated antigen-specific T cell responses as well as the production of protective antibodies with the ability to eliminate transformed or infected cells. More importantly, the vaccine composition may include two or more mRNAs coding for different proteins or long peptides. This enables the induction of polyclonal immune responses against a broad variety of epitopes within the encoded antigens that are presented on various MHC complexes, thus avoiding the restriction to a certain HLA molecule or possible immune escape due to antigen-loss. The development and design of mRNA therapies was recently boosted by several critical innovations including the development of technologies for the production and delivery of high quality and stable mRNA. Several technical obstacles such as stability, delivery and immunogenicity were addressed in the past and gradually solved in the recent years.This review will summarize the most recent technological developments and application of mRNA vaccines in clinical trials and discusses the results, challenges and future directions with a special focus on the induced innate and adaptive immune responses.
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http://dx.doi.org/10.1186/s12943-021-01339-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957288PMC
March 2021

BG-flow, a new flow cytometry tool for G-quadruplex quantification in fixed cells.

BMC Biol 2021 Mar 11;19(1):45. Epub 2021 Mar 11.

Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Venusberg-Campus 1, 53127, Bonn, Germany.

Background: Nucleic acids can fold into non-canonical secondary structures named G-quadruplexes (G4s), which consist of guanine-rich sequences stacked into guanine tetrads stabilized by Hoogsteen hydrogen bonding, π-π interactions, and monovalent cations. G4 structure formation and properties are well established in vitro, but potential in vivo functions remain controversial. G4s are evolutionarily enriched at distinct, functional genomic loci, and both genetic and molecular findings indicate that G4s are involved in multiple aspects of cellular homeostasis. In order to gain a deeper understanding of the function of G4 structures and the trigger signals for their formation, robust biochemical methods are needed to detect and quantify G4 structures in living cells. Currently available methods mostly rely on fluorescence microscopy or deep sequencing of immunoprecipitated DNA or RNA using G4-specific antibodies. These methods provide a clear picture of the cellular or genomic localization of G4 structures but are very time-consuming. Here, we assembled a novel protocol that uses the G4-specific antibody BG4 to quantify G4 structures by flow cytometry (BG-flow).

Results: We describe and validate a flow cytometry-based protocol for quantifying G4 levels by using the G4-specific antibody BG4 to label standard cultured cells (Hela and THP-1) as well as primary cells obtained from human blood (peripheral blood mononuclear cells (PBMCs)). We additionally determined changes in G4 levels during the cell cycle in immortalized MCF-7 cells, and validated changes previously observed in G4 levels by treating mouse macrophages with the G4-stabilizing agent pyridostatin (PDS).

Conclusion: We provide mechanistic proof that BG-flow is working in different kinds of cells ranging from mouse to humans. We propose that BG-flow can be combined with additional antibodies for cell surface markers to determine G4 structures in subpopulations of cells, which will be beneficial to address the relevance and consequences of G4 structures in mixed cell populations. This will support ongoing research that discusses G4 structures as a novel diagnostic tool.
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http://dx.doi.org/10.1186/s12915-021-00986-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953821PMC
March 2021

Elotuzumab spares dendritic cell integrity and functionality.

J Cancer Res Clin Oncol 2021 Mar 2. Epub 2021 Mar 2.

Medical Clinic III for Oncology, Hematology, Immune-Oncology and Rheumatology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany.

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http://dx.doi.org/10.1007/s00432-021-03572-zDOI Listing
March 2021

G-quadruplexes: a promising target for cancer therapy.

Mol Cancer 2021 02 25;20(1):40. Epub 2021 Feb 25.

Department of Oncology, Hematology, Rheumatology and Immune-Oncology, University Hospital Bonn, 53127, Bonn, Germany.

DNA and RNA can fold into a variety of alternative conformations. In recent years, a particular nucleic acid structure was discussed to play a role in malignant transformation and cancer development. This structure is called a G-quadruplex (G4). G4 structure formation can drive genome instability by creating mutations, deletions and stimulating recombination events. The importance of G4 structures in the characterization of malignant cells was currently demonstrated in breast cancer samples. In this analysis a correlation between G4 structure formation and an increased intratumor heterogeneity was identified. This suggests that G4 structures might allow breast cancer stratification and supports the identification of new personalized treatment options. Because of the stability of G4 structures and their presence within most human oncogenic promoters and at telomeres, G4 structures are currently tested as a therapeutic target to downregulate transcription or to block telomere elongation in cancer cells. To date, different chemical molecules (G4 ligands) have been developed that aim to target G4 structures. In this review we discuss and compare G4 function and relevance for therapeutic approaches and their impact on cancer development for three cancer entities, which differ significantly in their amount and type of mutations: pancreatic cancer, leukemia and malignant melanoma. G4 structures might present a promising new strategy to individually target tumor cells and could support personalized treatment approaches in the future.
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http://dx.doi.org/10.1186/s12943-021-01328-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905668PMC
February 2021

Vemurafenib as bridging therapy of hairy cell leukemia in a Jehovah's Witness patient.

Ann Hematol 2021 Jan 12. Epub 2021 Jan 12.

Medical Clinic III for Oncology, Hematology, Immune-Oncology and Rheumatology, University Hospital Bonn, Venusberg Campus 1, 53127, Bonn, Germany.

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http://dx.doi.org/10.1007/s00277-021-04403-4DOI Listing
January 2021

Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Nat Immunol 2020 05 23;21(5):555-566. Epub 2020 Apr 23.

Institute of Molecular Immunology, School of Life Sciences, TUM, Munich, Germany.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8 T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8 T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.
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http://dx.doi.org/10.1038/s41590-020-0666-9DOI Listing
May 2020

The role of checkpoint blockade after allogeneic stem cell transplantation in diseases other than Hodgkin's Lymphoma.

Bone Marrow Transplant 2019 10 4;54(10):1662-1667. Epub 2019 Mar 4.

Department of Hematology, Oncology and Rheumatology, University Hospital Bonn, Bonn, Germany.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment option for many malignant high-risk hematological diseases. The Graft-vs.-Tumor (GvT) effect is the major hallmark of this treatment approach. However, disease relapse remains a major limitation. Boosting the GvT effect by checkpoint inhibitors (CI) is an attractive option in this desperate situation although potentially triggering Graft-vs.-Host Disease (GvHD). Early reports in patients with Hodgkin's lymphoma support the idea that CI therapy after HSCT is feasible and effective. We have retrospectively analyzed CI therapy for treatment of disease recurrence after allo-HSCT other than Hodgkin's lymphoma including 21 patients from eight German transplant centers. The median follow-up was 59 days. The overall response rate (ORR) was 43%. Patients receiving donor lymphocyte infusion (DLI) in combination with CI had superior response (ORR 80%). Severe acute GvHD grade III-IV and moderate to severe chronic GvHD were observed in 29% of all patients. Taken together, CI therapy in relapsed patients after HSCT, especially in combination with DLI, is effective but induces severe GvHD in a considerable proportion of patients. Thus, prospective trials or EBMT registry-based validation of different dosing and application schedules including immunosuppressive regimens in those patients are urgently needed.
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http://dx.doi.org/10.1038/s41409-019-0498-0DOI Listing
October 2019

Successful Treatment of Refractory Squamous Cell Cancer of the Head and Neck with Nivolumab and Ipilimumab.

Case Rep Oncol 2018 Jan-Apr;11(1):17-20. Epub 2018 Jan 4.

aDepartment of Internal Medicine 3, University Hospital Bonn, Bonn, Germany.

Treatment options for patients with platinum-refractory, recurrent, metastatic head and neck squamous cell carcinoma (HNSCC) are limited, and prognosis is poor. Nivolumab (Opdivo) has been approved by the US Food and Drug Administration (FDA) for the treatment of patients with recurrent or metastatic HNSCC who have disease progression on or after platinum-based therapy. Recently, in patients with metastatic malignant melanoma a significant improvement of outcome and response was achieved with the combination of ipilimumab (CTLA4 antibody) and the programmed death (PD)-1 inhibitor nivolumab compared with monotherapy. Based on these results, the combination of nivolumab and ipilimumab has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma. So far, there have been no data concerning the combination of nivolumab and ipilimumab in squamous cell head and neck cancer. We here present the case of a 46-year-old male with refractory squamous cell head and neck cancer, who was successfully treated with the PD-1 inhibitor nivolumab in combination with the anti-CTLA4 antibody ipilimumab.
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http://dx.doi.org/10.1159/000485562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836192PMC
January 2018

Prolonged IKKβ Inhibition Improves Ongoing CTL Antitumor Responses by Incapacitating Regulatory T Cells.

Cell Rep 2017 Oct;21(3):578-586

Institute of Experimental Immunology, Rheinische Friedrich-Wilhelms-Universität, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany. Electronic address:

Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-κB signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-κB signaling through IκB-kinase β (IKKβ) after thymic egress. Mice lacking IKKβ in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3 Tregs. Also, pharmacological IKKβ inhibition reduced Treg numbers in the circulation by ∼50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKKβ inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKKβ inhibition after CTL cross-priming improved the antitumor response and delayed tumor growth. In conclusion, prolonged IKKβ inhibition decimates circulating Tregs and improves CTL responses when commenced after tumor vaccination, indicating that IKKβ represents a druggable checkpoint.
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http://dx.doi.org/10.1016/j.celrep.2017.09.082DOI Listing
October 2017

Targeting myeloid derived suppressor cells with all-trans retinoic acid is highly time-dependent in therapeutic tumor vaccination.

Oncoimmunology 2017;6(8):e1338995. Epub 2017 Jun 16.

Institute of Molecular Medicine, University Bonn, Germany.

Tumor immune escape is a critical problem which frequently accounts for the failure of therapeutic tumor vaccines. Among the most potent suppressors of tumor immunity are myeloid derived suppressor cells (MDSCs). MDSCs can be targeted by all-trans-retinoic-acid (atRA), which reduced their numbers and increased response rates in several vaccination studies. However, not much is known about the optimal administration interval between atRA and the vaccine as well as about its mode of action. Here we demonstrate in 2 different murine tumor models that mice unresponsive to a therapeutic vaccine harbored higher MDSC numbers than did responders. Application of atRA overcame MDSC-mediated immunosuppression and restored tumor control. Importantly, atRA was protective only when administered 3 d after vaccination (delayed treatment), whereas simultaneous administration even decreased the anti-tumor immune response and reduced survival. When analyzing the underlying mechanisms, we found that delayed, but not simultaneous atRA treatment with vaccination abrogated the suppressive capacity in monocytic MDSCs and instead caused them to upregulate MHC-class-II. Consistently, MDSCs from patients with colorectal carcinoma also failed to upregulate HLA-DR after treatment with TLR-ligation. Overall, we demonstrate that atRA can convert non-responders to responders to vaccination by suppressing MDSCs function and not only by reducing their number. Moreover, we identify a novel, strictly time-dependent mode of action of atRA to be considered during immunotherapeutic protocols in the future.
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http://dx.doi.org/10.1080/2162402X.2017.1338995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593699PMC
June 2017

Dexamethasone induced inhibition of Dectin-1 activation of antigen presenting cells is mediated via STAT-3 and NF-κB signaling pathways.

Sci Rep 2017 07 3;7(1):4522. Epub 2017 Jul 3.

Medical Clinic III for Oncology, Hematology, Immuno-Oncology and Rheumatology, University Hospital Bonn Sigmund-Freud-Straße 25, 53127, Bonn, Germany.

Treatment of patients with glucocorticoids can result in an increased risk of infection with pathogens such as fungi. Dectin-1 is a member of the C-type lectin receptor superfamily and was shown to be one of the major receptors for fungal beta-glucans. Activation of Dectin-1 increases the production of cytokines and chemokines and T-cell stimulatory capacity of DC and mediates resolution of fungal infections. Here we show that antigen-presenting cells generated in the presence of dexamethasone (Dex-DC) have a reduced capacity to stimulate T-cell proliferation and decreased expression of costimulatory molecules, that can not be enhanced upon stimulation with Dectin-1 ligands. Stimulation of Dex-DC with beta-glucans induced a strong upregulation of Syk phosphorylation and increased secretion of IL-10, while the production of IL-12, IL-23 and TNF-alpha was reduced. Downstream of Syk stimulation of Dectin-1 on Dex-DC resulted in phosphorylation of STAT3 and reduced nuclear localization of transcription factors involved in DC activation and function.
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http://dx.doi.org/10.1038/s41598-017-04558-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495798PMC
July 2017

Generation and functional characterization of MDSC-like cells.

Oncoimmunology 2017;6(4):e1295203. Epub 2017 Feb 23.

Medical Clinic III for Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany.

Myeloid-derived suppressor cells (MDSC) are critical in regulating immune responses by suppressing antigen presenting cells (APC) and T cells. We previously observed that incubation of peripheral blood monocytes with interleukin (IL)-10 during their differentiation to monocyte-derived dendritic cells (moDCs) results in the generation of an APC population with a CD14HLA-DRphenotype (IL-10-APC) with reduced stimulatory capacity similar to human MDSC. Co-incubation experiments now revealed that the addition of IL-10-APC to moDC caused a reduction of DC-induced T-cell proliferation, of the expression of maturation markers, and of secreted cytokines and chemokines such as TNF-α, IL-6, MIP-1α and Rantes. Addition of IL-10-APC increased the immunosuppressive molecule osteoactivin and its corresponding receptor syndecan-4 on moDC. Moreover, CD14HLA-DR MDSC isolated from healthy donors expressed high levels of osteoactivin, which was even further upregulated by the auxiliary addition of IL-10. Using transcriptome analysis, we identified a set of molecules and pathways mediating these effects. In addition, we found that IL-10-APC as well as human isolated MDSC expressed higher levels of programmed death (PD)-1, PD-ligand-1 (PD-L1), glucocorticoid-induced-tumor-necrosis-factor-receptor-related-protein (GITR) and GITR-ligand. Inhibition of osteoactivin, syndecan-4, PD-1 or PD-L1 on MDSC by using blocking antibodies restored the stimulatory capacity of DC in co-incubation experiments. Activation of MDSC with Dectin-1 ligand curdlan reduced the expression of osteoactivin and PD-L1. Our results demonstrate that osteoactivin/syndecan-4 and PD-/PD-L1 are key molecules that are profoundly involved in the inhibitory effects of MDSC on DC function and might be promising tools for clinical application.
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http://dx.doi.org/10.1080/2162402X.2017.1295203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414884PMC
February 2017

Variable resistance to freezing and thawing of CD34-positive stem cells and lymphocyte subpopulations in leukapheresis products.

Cytotherapy 2016 10 1;18(10):1325-31. Epub 2016 Aug 1.

Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn (UKB), Bonn, Germany. Electronic address:

Background Aims: Leukapheresis products for hematopoietic stem cell transplantation can be cryopreserved for various indications. Although it is known that CD34(+) cells tolerate cryopreservation well, a significant loss of CD3(+) cells has been observed, which has been ascribed to several factors, including transport, storage conditions and granulocyte-colony stimulating factor (G-CSF) administration.

Methods: To assess the tolerance of CD34(+) cells and lymphocyte subpopulations for cryopreservation and thawing, the post-thaw recoveries of CD34(+) cells, CD3(+)CD4(+) cells, CD3(+)CD8(+) cells, CD19(+) cells and CD16(+)CD56(+) cells were determined in 90 cryopreserved apheresis products, among which 65 were from G-CSF-mobilized donors, and 34 from unrelated donors that underwent transport before cryopreservation at our center. A controlled rate freezer and 5% dimethyl sulfoxide were used for cryopreservation.

Results: We could detect statistically significant differences for CD34(+) cell recovery (93.0 ± 20.7%) when compared to CD3(+)CD4(+) cell (83.1 ± 15.4%, P = 0.014), and CD3(+)CD8(+) cell recovery (83.3 ± 13.9%, P = 0.001). Similarly, CD19(+) cell recovery (98.6 ± 15.1%) was higher than CD3(+)CD4(+) cell (P = 2.5 × 10(-7)) and CD3(+)CD8(+) cell recovery (P = 1.2 × 10(-8)). Post-thaw recovery rates of all cell populations were not impaired in G-CSF-mobilized products compared with non-mobilized products nor in unrelated compared with related donor products.

Discussion: Our data suggest a lower tolerance of CD3(+) cells for cryopreservation and demonstrate that freezing-thawing resistance thawing is cell-specific and independent from other factors that affect post-thaw recovery of cryopreserved cells. Thus, a clinical consequence may be the monitoring of post-thaw CD3(+) cell doses of cryopreserved products, such as donor lymphocyte infusions.
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http://dx.doi.org/10.1016/j.jcyt.2016.06.014DOI Listing
October 2016

Long-term survival correlates with immunological responses in renal cell carcinoma patients treated with mRNA-based immunotherapy.

Oncoimmunology 2016 May 29;5(5):e1108511. Epub 2015 Oct 29.

Department of Hematology and Oncology, University of Bonn , Bonn, Germany.

Renal cell carcinoma (RCC) is an immunogenic tumor for which immunotherapeutic approaches could be associated with clinically relevant responses. It was recently shown, that induction of T-cell responses against multiple tumor-associated antigen (TAA) epitopes results in prolonged overall survival in RCC patients. In 2003-2005, we performed a phase I/II trial testing an mRNA-based vaccine formulation consisting of a mixture of in vitro transcribed RNA coding for six different TAAs (MUC1, CEA, Her2/neu, telomerase, survivin, MAGE-A1) in 30 metastatic RCC (mRCC) patients. In the first 14 patients, vaccinations were applied i.d. on days 0, 14, 28, and 42. In the consecutive 16 patients, an intensified protocol consisting of i.d. injections (daily on days 0-3, 7-10, 28, and 42) was used. After the respective induction periods, patients in both cohorts were vaccinated monthly until tumor progression. At survival update performed in July 2015, one of the 30 patients was still alive. One patient was lost to follow-up. Median survival of 24.5 mo (all patients) and 89 mo (favorable risk patients) exceeded predicted survival according to Memorial Sloan Kettering Cancer Center (MSKCC) risk score. Impressively, long-term survivors displayed immunological responses to the applied antigens while vice versa no patient without detectable immune response had survived more than 33 mo. The current survival update shows a clear correlation between survival and immunological responses to TAAs encoded by the naked mRNA vaccine. This is one of the first vaccination studies and the only RNA trial that reports on safety and efficacy after a follow-up of more than 10 y.
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http://dx.doi.org/10.1080/2162402X.2015.1108511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910748PMC
May 2016

γ-Glutamyl Transferase Is an Independent Biomarker of Splanchnic Thrombosis in Patients With Myeloproliferative Neoplasm.

Medicine (Baltimore) 2016 May;95(20):e3355

From the Department of Internal Medicine I (GJ, HLM, PM, JC, LJ, SC, TJ); Department of Medical Clinic III, University of Bonn, Bonn (VM, HA, BP, WD); Department of Hematology, Oncology, Hemostaseology, and SCT, Faculty of Medicine, University Hospital of the RWTH Aachen University, Aachen (KA, BTH, KS); Department of Biometrics, Informatics and Epidemiology, University of Bonn, Bonn (FR); Department for Hematology, University Hospital Essen, Essen (GJ); Department for Hematology, Oncology and Clinical Immunology, University Hospital Duesseldorf, Duesseldorf (GN); Practice for Hematology-Oncology Eppendorf, Hamburg (GE); and Department for Hematology, University Hospital Dresden, Dresden, Germany (PU).

Myeloproliferative neoplasms (MPNs) are associated with an increased risk of thrombotic events and constitute the major risk factor of splanchnic venous thrombosis (SVT) in Western countries. Although timely anticoagulation resolves SVT, unrecognized SVT frequently leads to portal hypertension and, potentially, variceal bleeding, which may render anticoagulation difficult. Thus, early identification of SVT development is clinically relevant in MPN patients.In this retrospective analysis, we included 126 patients with MPN and/or SVT referred to our hospital between 2009 and 2014. A total of 86 patients diagnosed with MPN formed the first cohort (PV n = 18, ET n = 16, and MF n = 40), whereas 40 patients who had SVT without adjunct MPN formed a control cohort. Median follow-up period was 960 days. Clinical and laboratory data were collected and analyzed for the identification of potential biomarkers applying descriptive statistics, nonparametric testing, Kaplan-Meier, and logistic regression analysis. The relevance of the identified biomarkers was evaluated in an independent 2nd cohort of 181 patients from the MPN registry of the Study Alliance of Leukemia (SAL-MPN).Thirty-three MPN patients (38%) in the 1st cohort had SVT. Elevated levels of aspartate aminotransferase, alanine aminotransferase, serum bilirubin, or γ-GT were significantly correlated to the presence of SVT. In multivariate testing, CRP and aspartate aminotransferase were predictors for survival and γ-GT remained the only significant variable associated with SVT in MPN patients (P < 0.05). These findings were confirmed in the 2nd cohort comprising 42% of patients with MPN suffering from SVT.Elevated γ-GT levels indicate SVT in MPN patients, whereas CRP levels are independent predictors of patient survival.
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http://dx.doi.org/10.1097/MD.0000000000003355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902387PMC
May 2016

Interferon gamma modulates sensitivity of CML cells to tyrosine kinase inhibitors.

Oncoimmunology 2016;5(1):e1065368. Epub 2015 Jul 1.

Department of Oncology, Hematology and Rheumatology, University Hospital Bonn , Bonn, Germany.

Immune effector cells such as T and NK cells can efficiently eliminate tumor cells. However, when activating oncogenic signaling pathways or protective mechanisms against cell death are active, immune cells can also confer therapy resistance. Here, we analyzed the role of activated T and NK cells and released cytokines on tyrosine kinase inhibitors imatinib and nilotinib - mediated apoptosis induction and proliferation of chronic myelogenous leukemia (CML) cells. Incubation of CML cells with activated, but not with resting CD3 T cells or with activated NK cells significantly inhibited TKI-induced apoptosis induction in CML cells as quantified by nuclear fragmentation assays. Transwell experiments revealed a critical role for T or NK cell-derived cytokines for CML cell protection. Accordingly, CML cells treated with IFNγ also showed a clearly reduced sensitivity to TKI-mediated cell death induction and inhibition of proliferation. In contrast, IFNα or other pro-inflammatory mediators and cytokines, such as TNFα and GM-CSF did not impair TKI-induced apoptosis in CML cells. On a molecular level, IFNγ-exposed CML cells showed a significantly reduced caspase-3 activation and PARP-1 cleavage as well as an increased expression of anti-apoptotic molecule xIAP. Finally, IFNγ diminished TKI-induced downregulation of Jak-2 and STAT-5 phosphorylation and increased nuclear expression of RUNX-1, which may at least in part contribute to the reduced sensitivity to TKI effects. Our results demonstrate that IFNγ released by activated T or NK cells may interfere with the therapeutic effects of TKI in CML. Our findings may have important implications for the understanding of inflammation-mediated BCR-ABL independent resistance mechanisms.
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http://dx.doi.org/10.1080/2162402X.2015.1065368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760295PMC
July 2015

The induction of human myeloid derived suppressor cells through hepatic stellate cells is dose-dependently inhibited by the tyrosine kinase inhibitors nilotinib, dasatinib and sorafenib, but not sunitinib.

Cancer Immunol Immunother 2016 Mar 19;65(3):273-82. Epub 2016 Jan 19.

Institute of Molecular Medicine, University Bonn, Sigmund-Freud-Straße 25, 53127, Bonn, Germany.

Increased numbers of immunosuppressive myeloid derived suppressor cells (MDSCs) correlate with a poor prognosis in cancer patients. Tyrosine kinase inhibitors (TKIs) are used as standard therapy for the treatment of several neoplastic diseases. However, TKIs not only exert effects on the malignant cell clone itself but also affect immune cells. Here, we investigate the effect of TKIs on the induction of MDSCs that differentiate from mature human monocytes using a new in vitro model of MDSC induction through activated hepatic stellate cells (HSCs). We show that frequencies of monocytic CD14(+)HLA-DR(-/low) MDSCs derived from mature monocytes were significantly and dose-dependently reduced in the presence of dasatinib, nilotinib and sorafenib, whereas sunitinib had no effect. These regulatory effects were only observed when TKIs were present during the early induction phase of MDSCs through activated HSCs, whereas already differentiated MDSCs were not further influenced by TKIs. Neither the MAPK nor the NFκB pathway was modulated in MDSCs when any of the TKIs was applied. When functional analyses were performed, we found that myeloid cells treated with sorafenib, nilotinib or dasatinib, but not sunitinib, displayed decreased suppressive capacity with regard to CD8+ T cell proliferation. Our results indicate that sorafenib, nilotinib and dasatinib, but not sunitinib, decrease the HSC-mediated differentiation of monocytes into functional MDSCs. Therefore, treatment of cancer patients with these TKIs may in addition to having a direct effect on cancer cells also prevent the differentiation of monocytes into MDSCs and thereby differentially modulate the success of immunotherapeutic or other anti-cancer approaches.
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http://dx.doi.org/10.1007/s00262-015-1790-5DOI Listing
March 2016

Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma.

Exp Hematol Oncol 2015 5;4:21. Epub 2015 Aug 5.

Medical Clinic III, Department of Hematology and Oncology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.

Background: Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM.

Methods: The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235).

Results: Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.

Conclusions: Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.
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http://dx.doi.org/10.1186/s40164-015-0016-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526421PMC
August 2015

The VEGF-Receptor Inhibitor Axitinib Impairs Dendritic Cell Phenotype and Function.

PLoS One 2015 4;10(6):e0128897. Epub 2015 Jun 4.

Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany.

Inhibitors of VEGF receptor (VEGFR) signaling such as sorafenib and sunitinib that are currently used in the treatment of malignant diseases have been shown to affect immunological responses by inhibition of the function of antigen presenting cells and T lymphocytes. The VEGFR-inhibitor axitinib has recently been approved for second line therapy of metastatic renal cell carcinoma. While there is some evidence that axitinib might interfere with the activation of T cells, not much is known about the effects of axitinib on dendritic cell (DC) phenotype and function. We here show that the addition of axitinib during the final Toll-like receptor-4-induced maturation step of monocyte-derived human DCs results in a reduced DC activation characterized by impaired expression of activation markers and co-stimulatory molecules such as CD80, CD83 and CD86. We further found a decreased secretion of interleukin-12 which was accompanied by reduced nuclear expression of the transcription factor cRel. In addition, we found a dose-dependent reduced activation of p38 and STAT3 in axitinib-exposed DCs, whereas the expression was not affected. The dysfunction of axitinib-exposed DCs was further underlined by their impaired induction of allogeneic T cell proliferation in a mixed lymphocyte reaction assay and inhibition of DC migration. Our results demonstrate that axitinib significantly affects DC differentiation and function primarily via the inhibition of the nuclear factor kappa B signaling pathway leading to impaired T cell activation. This will be of importance for the design of future vaccination protocols and therapeutic approaches aiming at combining different treatment strategies, eg such as programmed death-1 inhibitors with axitinib.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456373PMC
May 2016

A party of three: iNKT cells in GVHD prevention.

Blood 2015 May;125(22):3374-5

UNIVERSITY HOSPITAL BONN.

In this issue of Blood, Schneidawind et al demonstrate that the adoptive transfer of CD4+ invariant natural killer T (iNKT) cells from third-party mice protects from lethal graft-versus-host disease (GVHD) through expansion of donor regulatory T cells (Tregs) in a murine model of allogeneic hematopoietic cell transplantation (HCT).
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http://dx.doi.org/10.1182/blood-2015-04-636282DOI Listing
May 2015

JAK Inhibition Impairs NK Cell Function in Myeloproliferative Neoplasms.

Cancer Res 2015 Jun 1;75(11):2187-99. Epub 2015 Apr 1.

Medical Clinic, Oncology, Hematology and Rheumatology, University Clinic Bonn (UKB), Bonn, Germany.

Ruxolitinib is a small-molecule inhibitor of the JAK kinases, which has been approved for the treatment of myelofibrosis, a rare myeloproliferative neoplasm (MPN), but clinical trials are also being conducted in inflammatory-driven solid tumors. Increased infection rates have been reported in ruxolitinib-treated patients, and natural killer (NK) cells are immune effector cells known to eliminate both virus-infected and malignant cells. On this basis, we sought to compare the effects of JAK inhibition on human NK cells in a cohort of 28 MPN patients with or without ruxolitinib treatment and 24 healthy individuals. NK cell analyses included cell frequency, receptor expression, proliferation, immune synapse formation, and cytokine signaling. We found a reduction in NK cell numbers in ruxolitinib-treated patients that was linked to the appearance of clinically relevant infections. This reduction was likely due to impaired maturation of NK cells, as reflected by an increased ratio in immature to mature NK cells. Notably, the endogenous functional defect of NK cells in MPN was further aggravated by ruxolitinib treatment. In vitro data paralleled these in vivo results, showing a reduction in cytokine-induced NK cell activation. Further, reduced killing activity was associated with an impaired capacity to form lytic synapses with NK target cells. Taken together, our findings offer compelling evidence that ruxolitinib impairs NK cell function in MPN patients, offering an explanation for increased infection rates and possible long-term side effects associated with ruxolitinib treatment.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-3198DOI Listing
June 2015

JAK1/2 inhibition impairs T cell function in vitro and in patients with myeloproliferative neoplasms.

Br J Haematol 2015 Jun 30;169(6):824-33. Epub 2015 Mar 30.

Department of Internal Medicine III, Oncology, Haematology and Rheumatology, University Hospital Bonn (UKB), Bonn, Germany.

Ruxolitinib (INCB018424) is the first JAK1/JAK2 inhibitor approved for treatment of myelofibrosis. JAK/STAT-signalling is known to be involved in the regulation of CD4(+) T cells, which critically orchestrate inflammatory responses. To better understand how ruxolitinib modulates CD4(+) T cell responses, we undertook an in-depth analysis of CD4(+) T cell function upon ruxolitinib exposure. We observed a decrease in total CD3(+) cells after 3 weeks of ruxolitinib treatment in patients with myeloproliferative neoplasms. Moreover, we found that the number of regulatory T cells (Tregs), pro-inflammatory T-helper cell types 1 (Th1) and Th17 were reduced, which were validated by in vitro studies. In line with our in vitro data, we found that inflammatory cytokines [tumour necrosis factor-α (TNF), interleukin (IL)5, IL6, IL1B] were also downregulated in T cells from patients (all P < 0·05). Finally, we showed that ruxolitinib does not interfere with the T cell receptor signalling pathway, but impacts IL2-dependent STAT5 activation. These data provide a rationale for testing JAK inhibitors in diseases triggered by hyperactive CD4(+) T cells, such as autoimmune diseases. In addition, they also provide a potential explanation for the increased infection rates (i.e. viral reactivation and urinary tract infection) seen in ruxolitinib-treated patients.
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http://dx.doi.org/10.1111/bjh.13373DOI Listing
June 2015

Implementing combinatorial immunotherapeutic regimens against cancer: The concept of immunological conditioning.

Oncoimmunology 2014 Jan 3;3(1):e27588. Epub 2014 Jan 3.

Medical Clinic III; Department of Oncology, Hematology and Rheumatology; University Hospital Bonn (UKB); Bonn, Germany.

Harnessing the host immune system to eradicate cancer has a high therapeutic potential. One paradigm of anticancer immunotherapy is represented by allogeneic stem cell transplantation. In this setting, the host must be conditioned prior to transplantation, allowing for engraftment and subsequent graft-vs.-tumor reactivity. Conditioning may also be a prerequisite for the efficacy of other immunotherapeutic regimens. In particular, tumor debulking followed by conditioning (aimed at blocking endogenous inhibitory stimuli, for instance upon the depletion of regulatory T cells or the inhibition of immune checkpoints) and subsequent immunization (for instance by means of patient-tailored vaccines) based on innovative adjuvants (such as RIG-I ligands) may allow for the elicitation of superior antitumor immune responses. Repetitive boosting might then maintain immunosurveillance. An intense wave of investigation on the optimal timing of immunostimulatory interventions with respect to the administration of immunogenic chemotherapeutics and on the use of small drugs that promote efficient antitumor immune responses will end up in the generation of highly effective immunotherapeutic anticancer regimens.
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http://dx.doi.org/10.4161/onci.27588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006858PMC
January 2014

Ruxolitinib is a potent immunosuppressive compound: is it time for anti-infective prophylaxis?

Blood 2013 Nov;122(23):3843-4

Medical Clinic III for Oncology, Haematology and Rheumatology, University Hospital Bonn, Bonn, Germany.

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http://dx.doi.org/10.1182/blood-2013-10-531103DOI Listing
November 2013

Advances in immunotherapy of chronic myeloid leukemia CML.

Curr Cancer Drug Targets 2013 Sep;13(7):768-74

Medizinische Klinik III, Hämatologie/Onkologie; Wilhelmstr. 35-37, 53111 Bonn in Sigmund-Freud- Str. 25, 53105 Bonn, Germany.

Tyrosine kinase inhibitors induce sustained disease remissions in chronic myeloid leukemia by exploiting the addiction of this type of leukemia to the activity of the fusion oncogene BCR-ABL. However, these agents fail to eradicate CML stem cells which are ultimately responsible for disease relapses upon treatment discontinuation. Evidence that the immune system can effectively reject CML stem cells potentially leading to patient cure is provided by the experience with patients receiving allogeneic bone marrow transplantations. Compelling evidence indicates that more modern, antigen-specific immunotherapeutic approaches are also feasible and hold strong potential to be clinically effective. Amongst these, particularly promising is the use of autologous dendritic cells pulsed with antigens or direct application of in vitro transcribed RNA encoding for leukemia-associated antigens, since this approach allows to circumvent HLA-restriction of the leukemia-associated T cell epitopes that have been eventually identified. Combining these strategies with monoclonal antibodies, such as anti-CTLA-4 or anti-PD-1, may help to obtain even stronger immune responses and better clinical results. This narrative review addresses this topic by focusing in particular on the cell-based immunotherapeutic strategies for CML and on the issue of the leukemia-associated antigens to be selected for targeting.
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http://dx.doi.org/10.2174/15680096113139990086DOI Listing
September 2013

The JAK-inhibitor ruxolitinib impairs dendritic cell function in vitro and in vivo.

Blood 2013 Aug 14;122(7):1192-202. Epub 2013 Jun 14.

Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany.

The Janus kinase (JAK)-inhibitor ruxolitinib decreases constitutional symptoms and spleen size of myelofibrosis (MF) patients by mechanisms distinct from its anticlonal activity. Here we investigated whether ruxolitinib affects dendritic cell (DC) biology. The in vitro development of monocyte-derived DCs was almost completely blocked when the compound was added throughout the differentiation period. Furthermore, when applied solely during the final lipopolysaccharide-induced maturation step, ruxolitinib reduced DC activation as demonstrated by decreased interleukin-12 production and attenuated expression of activation markers. Ruxolitinib also impaired both in vitro and in vivo DC migration. Dysfunction of ruxolitinib-exposed DCs was further underlined by their impaired induction of allogeneic and antigen-specific T-cell responses. Ruxolitinib-treated mice immunized with ovalbumin (OVA)/CpG induced markedly reduced in vivo activation and proliferation of OVA-specific CD8⁺ T cells compared with vehicle-treated controls. Finally, using an adenoviral infection model, we show that ruxolitinib-exposed mice exhibit delayed adenoviral clearance. Our results demonstrate that ruxolitinib significantly affects DC differentiation and function leading to impaired T-cell activation. DC dysfunction may result in increased infection rates in ruxolitinib-treated patients. However, our findings may also explain the outstanding anti-inflammatory and immunomodulating activity of JAK inhibitors currently used in the treatment of MF and autoimmune diseases.
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http://dx.doi.org/10.1182/blood-2013-03-484642DOI Listing
August 2013

Imatinib mesylate and nilotinib affect MHC-class I presentation by modulating the proteasomal processing of antigenic peptides.

Cancer Immunol Immunother 2013 Apr 25;62(4):715-26. Epub 2012 Nov 25.

Department of Hematology and Oncology, University Hospital Bonn, Bonn, Germany.

Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and -B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.
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http://dx.doi.org/10.1007/s00262-012-1373-7DOI Listing
April 2013

Regulation of dectin-1-mediated dendritic cell activation by peroxisome proliferator-activated receptor-gamma ligand troglitazone.

Blood 2011 Mar 4;117(13):3569-74. Epub 2011 Feb 4.

Department of Hematology and Oncology, University of Bonn, Wilhelmstrasse 35-37, Bonn, Germany.

Dectin-1 is the major receptor for fungal β-glucans. The activation of Dectin-1 leads to the up-regulation of surface molecules on dendritic cells (DCs) and cytokine secretion. Furthermore, Dectin-1 is important for the recruitment of leukocytes and the production of inflammatory mediators. Peroxisome proliferator-activated receptor-γ (PPAR-γ) and its ligands, cyclopentenone prostaglandins or thiazolidinediones, have modulatory effects on B-cell, T-cell, and DC function. In the present study, we analyzed the effects of troglitazone (TGZ), a high-affinity synthetic PPAR-γ ligand, on the Dectin-1-mediated activation of monocyte-derived human DCs. Dectin-1-mediated activation of DCs was inhibited by TGZ, as shown by down-regulation of costimulatory molecules and reduced secretion of cytokines and chemokines involved in T-lymphocyte activation. Furthermore, TGZ inhibited the T-cell-stimulatory capacity of DCs. These effects were not due to a diminished expression of Dectin-1 or to a reduced phosphorylation of spleen tyrosine kinase; they were mediated by the inhibition of downstream signaling molecules such as mitogen-activated protein kinases and nuclear factor-κB. Furthermore, curdlan-mediated accumulation of caspase recruitment domain 9 (CARD9) in the cytosol was inhibited by TGZ. Our data demonstrate that the PPAR-γ ligand TGZ inhibits Dectin-1-mediated activation by interfering with CARD9, mitogen-activated protein kinase, and nuclear factor-κB signaling pathways. This confirms their important role as negative-feedback regulators of potentially harmful inflammatory responses.
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http://dx.doi.org/10.1182/blood-2010-08-302224DOI Listing
March 2011